F. oxysporum f.sp. melonis race 1,2-resistant melons

ABSTRACT

Methods for conveying  Fusarium oxysporum  f.sp.  melonis  (FOM) race 1,2 resistance into non-resistant melon germplasm are provided, in some embodiments, the methods include introgressing FOM race 1,2 resistance into a non-resistant melon using one or more nucleic acid markers for marker-assisted selection among melon lines to be used in a melon breeding program, wherein the markers are linked to FOM race 1,2 resistance. Also provided are quantitative trait loci (QTLs) associated with resistance to FOM race 1,2; isolated and purified genetic markers associated with FOM race 1,2 resistance; melon plants, seeds, and tissue cultures produced by any of the disclosed methods; fruit and seed produced by the disclosed melon plants; and compositions including amplification primer pairs capable of initiating DNA polymerization by a DNA polymerase on melon nucleic acid templates to generate melon marker amplicons.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase application of InternationalApplication No. PCT/EP2008/057770, filed Jun. 19, 2008, which claimspriority to European Application No. 07110860.9, filed Jun. 22, 2007.

TECHNICAL FIELD

The presently disclosed subject matter relates to melons, such as melonsof the species Cucumis melo, and methods of breeding the same. Moreparticularly, the presently disclosed subject matter relates to melonlines, such as Cucumis melo lines, with improved resistance to. Fusariumoxysporum f.sp. melonis race 1,2 infection and methods of breeding same,the methods involving genetic marker analysis.

BACKGROUND

Plant pathogens are known to cause massive damage to important crops,resulting in significant agricultural losses with widespreadconsequences for both the food supply and other industries that rely onplant materials. As such, there is a long felt need to reduce theincidence and/or impact of agricultural pests on crop production.

An example of such pathogens is the Fusarium oxysporum genus of plantfungi. F. oxysporum is known to devastate various crop plants including,but not limited to pea, banana, cotton, tomato, and others. F. oxysporumis characterized by several different specialized forms, which arereferred to as formae specialis (f.sp.), each of which infect a varietyof hosts to cause disease. There are at least 48 different formaespeciales of F. oxysporum.

One particular formae special's of F. oxysporum is. F. oxysporum f.sp.melonis (FOM), which infects various melons of the species Cucumis melo,which includes European cantaloupes and includes muskmelons such asAmerican cantaloupes, sugar melons, honeydews, and Casaba. Several raceshave been identified for FOM, and include races 0, 1, 2, and 1,2.Additionally, two genes, Fom-1 and Fom-2, have been identified that areassociated with resistance to races 0 and 2, and 0 and 1, respectively(Risser et al., 1976).

What are needed, then, are new hybrid and/or inbred Cucumis melovarieties that are resistant to FOM race 1,2, and new methods forintroducing increased resistance to FOM race 1,2 in melons.

SUMMARY

This Summary lists several embodiments of the presently disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This Summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently disclosed subjectmatter, whether listed in this Summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

The presently disclosed subject matter provides methods for conveyingFusarium oxysporum f.sp. melonis (FOM) race 1,2 resistance intonon-resistant melon germplasm. In some embodiments the methods compriseintrogressing FOM race 1,2 resistance into a non-resistant melon usingone or more nucleic acid markers for marker-assisted selection amongmelon lines to be used in a melon breeding program, wherein the markersare linked to FOM race 1,2 resistance. In some embodiments, the one ormore nucleic acid marker is selected from the group consisting of 3.1,3.2, 6.1, 6.2, 7.1, 7.2, 9.1, 9.2, 9.3, 10.1, and 10.2. In someembodiments, the one or more nucleic acid markers are selected from thegroup consisting of 3.1, 3.2, 9.1, 9.2, and 9.3. In some embodiments,the marker-assisted selection comprises the use of an analysis techniqueselected from the group consisting of RAPD analysis, RFLP analysis,microsatellite analysis, and AFLP analysis. In some embodiments, themethods further comprise screening an introgressed melon for orangeflesh.

The presently disclosed subject matter also provides methods forreliably and predictably introgressing FOM race 1,2 resistance intonon-resistant melon germplasm. In some embodiments, the methods compriseusing one or more nucleic acid markers for marker-assisted selectionamong melon lines to be used in a melon breeding program, wherein thenucleic acid markers are selected from the group consisting of 3.1, 3.2,6.1, 6.2, 7.1, 7.2, 9.1, 9.2, 9.3, 10.1, and 10.2, and introgressing theresistance into the non-resistant melon germplasm. In some embodiments,the one or more nucleic acid markers are selected from the groupconsisting of 3.1, 3.2, 9.1, 9.2, and 9.3. In some embodiments, themarker-assisted selection comprises the use of an analysis techniqueselected from the group consisting of RAPD analysis, RFLP analysis,microsatellite analysis, and AFLP analysis. In some embodiments, themethods further comprise screening an introgressed melon for orangeflesh.

The presently disclosed subject matter also provides methods for theproduction of an inbred melon plant adapted for conferring, in hybridcombination with a suitable second inbred, resistance to F. oxysporumf.sp. melon's (FOM) race 1,2. In some embodiments, the methods comprise(a) selecting a first donor parental line possessing a desired FOM race1,2 resistance and having at least one of the resistant loci selectedfrom a locus mapping to linkage group 3 and mapped by one or more of themarkers 3.1 and 3.2 and a locus mapping to linkage group 9 and mapped byone or more of the markers 9.1, 9.2, and 9.3; (b) crossing the firstdonor parent line with a second parental line in hybrid combination, toproduce a segregating plant population; (c) screening the segregatingplant population for identified chromosomal loci of one or more genesassociated with the resistance to FOM race 1,2; and (d) selecting plantsfrom the population having the identified chromosomal loci for furtherscreening until a line is obtained which is homozygous for resistance toFOM race 1,2 at sufficient loci to give resistance to FOM race 1,2 inhybrid combination. In some embodiments, the methods further comprisescreening the plants of the line that is homozygous for resistance toFOM race 1,2 at sufficient loci to give resistance to FOM race 1,2 inhybrid combination for the presence of orange flesh. In someembodiments, the methods further comprise screening one or more membersof the segregating plant population of the selected plants for orangeflesh.

The presently disclosed subject matter also provides methods forproducing melon plants which are resistant to F. oxysporum f.sp. melonis(FOM) race 1,2 occurring in melon. In some embodiments, the methodscomprise (a) providing a Cucumis melo plant which contains one or morealleles that confer resistance to FOM race 1,2, which alleles arecharacterized one or more of five Quantitative Trait Loci QTL1-QTL5 ondifferent chromosomes, wherein (i) QTL1 is defined by the followingmarkers: (1) a marker of about 322 basepairs (bp), wherein the markercorresponds to an amplification product generated by amplifying aCucumis melo nucleic acid with a forward primer comprising a nucleotidesequence as set forth in SEQ ID NO: 1 and a reverse primer comprising anucleotide sequence as set forth in SEQ ID NO: 2; and (2) a marker ofabout 303 bp, wherein the marker corresponds to an amplification productgenerated by amplifying a Cucumis melo nucleic acid with a forwardprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 3 anda reverse primer comprising a nucleotide sequence as set forth in SEQ IDNO: 4, or any part of a DNA sequence as in 03MFR001795 linked within 1,2, 5, or 10 cM to at least one of the markers of (1) and (2) conferringresistance to FOM race 1,2; (ii) QTL2 is defined by the followingmarkers: (1) a marker of about 237 bp, wherein the marker corresponds toan amplification product generated by amplifying a Cucumis melo nucleicacid with a forward primer comprising a nucleotide sequence as set forthin SEQ ID NO: 5 and a reverse primer comprising a nucleotide sequence asset forth in SEQ ID NO: 6; and (2) a marker of about 189 bp, wherein themarker corresponds to an amplification product generated by amplifying aCucumis melo nucleic acid with a forward primer comprising a nucleotidesequence as set forth in SEQ ID NO: 7 and a reverse primer comprising anucleotide sequence as set forth in SEQ ID NO: 8, or any part of a DNAsequence as in 03MFR001795 linked within 1, 2, 5, or 10 cM to at leastone of the markers of (1) and (2) conferring resistance to FOM race 1,2;(iii) QTL3 is defined by the following markers: (1) a marker of about221 bp, wherein the marker corresponds to an amplification productgenerated by amplifying a Cucumis melo nucleic acid with a forwardprimer comprising a nucleotide sequence as set forth in SEQ ID NO: 9 anda reverse primer comprising a nucleotide sequence as set forth in SEQ IDNO: 10; and (2) a marker of about 229 bp, wherein the marker correspondsto an amplification product generated by amplifying a Cucumis melonucleic acid with a forward primer comprising a nucleotide sequence asset forth in SEQ ID NO: 11 and a reverse primer comprising a nucleotidesequence as set forth in SEQ ID NO: 12, or any part of a DNA sequence asin 03MFR001795 linked within 1, 2, 5, or 10 cM to at least one of themarkers of (1), and (2) conferring resistance to FOM race 1,2; (iv) QTL4is defined by the following markers: (1) a marker of about 329 bp,wherein the marker corresponds to an amplification product generated byamplifying a Cucumis melo nucleic acid with a forward primer comprisinga nucleotide sequence as set forth in SEQ ID NO: 13 and a reverse primercomprising a nucleotide sequence as set forth in SEQ ID NO: 14; (2) amarker of about 279 bp, wherein the marker corresponds to anamplification product generated by amplifying a Cucumis melo nucleicacid with a forward primer comprising a nucleotide sequence as set forthin SEQ ID NO: 15 and a reverse primer comprising a nucleotide sequenceas set forth in SEQ ID NO: 16; and (3) a marker of about 147 bp, whereinthe marker corresponds to an amplification product generated byamplifying a Cucumis melo nucleic acid with a forward primer comprisinga nucleotide sequence as set forth in SEQ ID NO: 17 and a reverse primercomprising a nucleotide sequence as set forth in SEQ ID NO: 18, or anypart of a DNA sequence as in 03MFR001795 linked within 1, 2, 5, or 10 cMto at least one of the markers of (1), (2), and (3) conferringresistance to FOM race 1,2; and (v) QTL5′ is defined by the followingmarkers: (1) a marker of about 246 bp, wherein the marker corresponds toan amplification, product generated by amplifying a Cucumis melo nucleicacid with a forward primer comprising a nucleotide sequence as set forthin SEQ ID NO: 1.9 and a reverse primer comprising a nucleotide sequenceas set forth in SEQ ID NO: 20; and (2) a marker of about 251 bp, whereinthe marker corresponds to an amplification product generated byamplifying a Cucumis melo nucleic acid with a forward primer comprisinga nucleotide sequence as set forth in SEQ ID NO: 21 and a reverse primercomprising a nucleotide sequence as set forth in SEQ ID NO: 22, or anypart of a DNA sequence as in 03MFR001795 linked within 1, 2, 5, or 10 cMto at least one of the markers of (1) and (2) conferring resistance toFOM race 1,2; (b) crossing the Cucumis melo plant provided in step (a)with Cucumis melo culture breeding material to produce one or moreprogeny individuals, whereby one or more melon plants which areresistant to F. oxysporum f.sp. melonis (FOM) race 1,2 occurring inmelon are produced. In some embodiments, the methods further comprise(c) collecting the seeds resulting from the cross in step (b); (d)regenerating the seeds into plants; (e) evaluating the plants of step(d) for resistance to FOM; and (f) identifying and selecting plantswhich are resistant to the FOM race 1,2. In some embodiments, theCucumis melo plant provided in step (a) is 03MFR001795. In someembodiments, the FOM race 1,2 strain is a yellowing strain.

The presently disclosed subject matter also provides methods forproducing seeds that result in melon plants resistant to FOM race 1,2occurring in melon. In some embodiments, the methods comprise (a)providing a Cucumis melo plant which contains one or more alleles thatconfer resistance to FOM race 1,2, which alleles are characterized byone or more of five Quantitative Trait Loci QTL1-QTL5 on differentchromosomes, wherein QTL1-QTL5 are as defined herein; (b) crossing theCucumis melo plant provided in step (a) with Cucumis melo culturebreeding material; and (c) collecting seeds resulting from the cross instep (b) that result in melon plants which are resistant to FOM,particularly FOM race 1,2.

The presently disclosed subject matter also provides methods foridentifying a first Cucumis melo plant or germplasm that displaysresistance, improved resistance, or reduced susceptibility to FOM race1,2. In some embodiments, the methods comprise detecting in the firstCucumis melo plant or germplasm at least one allele of one or moremarker locus that is associated with the resistance, improvedresistance, or reduced susceptibility, wherein the one or more markerlocus is selected from the group consisting of (a) 3.1, 3.2, 6.1, 6.2,7.1, 7.2, 9.1, 9.2, 9.3, 10.1, and 10.2; (b) a marker locus linked to amarker locus of (a); and (c) a marker locus localizing within achromosome interval including a marker pair selected from the groupconsisting of 3.1 and 3.2, 6.1 and 6.2, 7.1 and 7.2, 9.1 and 9.3, and10.1 and 10.2. In some embodiments, the closely linked marker locus of(b) displays a genetic recombination frequency of less than about 10%,optionally, less than 5%, and further optionally less than about 1%,with the marker locus of (a). In some embodiments, the one or moremarker locus associated with resistance, improved resistance, or reducedsusceptibility is selected from the marker loci of (a) and (b). In someembodiments, the one or more marker locus associated with resistance,improved resistance, or reduced susceptibility is a plurality of lociselected from the marker loci of (a) and (b). In some embodiments, theone or more marker locus associated with resistance, improvedresistance, or reduced susceptibility is selected from marker locilocalizing within the chromosome intervals of (c). In some embodiments,the one or more marker locus associated with resistance, improvedresistance, or reduced susceptibility is a plurality of loci selectedfrom marker loci localizing within the chromosome intervals of (c). Insome embodiments, the germplasm is a Cucumis melo line or variety. Insome embodiments, the resistance, improved resistance, or reducedsusceptibility to FOM race 1,2 is assayed in a field location previouslyknown to produce Cucumis melo plants that demonstrate FOM race 1,2infection. In some embodiments, the FOM race 1,2 is a yellowing strain.In some embodiments, the resistance, improved resistance, or reducedsusceptibility further provides resistance, improved resistance, orreduced susceptibility to at least one of FOM races 0, 1, and 2. In someembodiments, the detecting comprises detecting at least one allelic formof a polymorphic simple sequence repeat (SSR) or a single nucleotidepolymorphism (SNP). In some embodiments, the detecting comprisesamplifying the marker, locus or a portion of the marker locus anddetecting the resulting amplified marker amplicon. In some embodiments,the amplifying comprises: (a) admixing an amplification primer oramplification primer pair with a nucleic acid isolated from the firstCucumis melo plant or germplasm, wherein the primer or primer pair iscomplementary or partially complementary to at least a portion of themarker locus, and is capable of initiating DNA polymerization by a DNApolymerase using the melon nucleic acid as a template; and (b) extendingthe primer or primer pair in a DNA polymerization reaction comprising aDNA polymerase and a template nucleic acid to generate at least oneamplicon. In some embodiments, the nucleic acid is selected from DNA andRNA. In some embodiments, the at least one allele is an SNP allele, themethod comprising, detecting the SNP using allele specific hybridization(ASH) analysis. In some embodiments, the amplifying comprises employinga polymerase chain reaction (PCR) or ligase chain reaction (LCR) using anucleic acid isolated from the first melon plant or germplasm as atemplate in the PCR or LCR. In some embodiments, the at least one alleleis a favorable allele that positively correlates with resistance,improved resistance, or reduced susceptibility. In some embodiments, theat least one allele comprises two or more alleles. In some embodiments,the at least one allele is correlated with resistance, improvedresistance, or reduced susceptibility to FOM race 1,2, the methodcomprising introgressing the allele in the first Cucumis melo plant orgermplasm into a second Cucumis melo plant or germplasm to produce anintrogressed Cucumis melo plant or germplasm. In some embodiments, thesecond Cucumis melo plant or germplasm displays less resistance to FOMrace 1,2 infection as compared to the first Cucumis melo plant orgermplasm, and wherein the introgressed Cucumis melo plant or germplasmdisplays an increased resistance, improved resistance, or reducedsusceptibility to FOM race 1,2 infection as compared to the secondCucumis melo plant or germplasm.

The presently disclosed subject matter also provides quantitative traitloci (QTLs) associated with resistance to FOM race 1,2 in a melon. Insome embodiments, the QTLs map to a linkage group in the melon genomeselected from linkage group 3 mapped by one or more of the markers 3.1,3.2, 3.3, and 3.4; linkage group 6 and mapped by one or more of themarkers 6.1, 6.2, and 6.3; linkage group 7 and mapped by one or more ofthe markers 7.1, 7.2, and 7.3; linkage group 9 and mapped by one or moreof the markers 9.1, 9.2, and 9.3; and linkage group 10 and mapped by oneor more of the markers 10.1, 10.2, and 10.3.

The presently disclosed subject matter also provides isolated andpurified genetic markers associated with FOM race 1,2 resistance inCucumis melo. In some embodiments, the markers (a) map to a linkagegroup in a Cucumis melo genome, the linkage group selected from thegroup consisting of QTL1, QTL2, QTL3, QTL4, and QTL5; or (b) areselected from the group consisting of 3.1, 3.2, 6.1, 6.2, 7.1, 7.2, 9.1,9.2, 9.3, 10.1, and 10.2; or (c) comprise a nucleotide sequence selectedof at least 10 contiguous bases, optionally at least 15 contiguousbases, and also optionally the full length sequence of any of odd SEQ IDNOs: 1-21 or the complement of any of even SEQ ID NOs: 2-22; or (d)comprise a nucleotide sequence of at least 10 contiguous nucleotidescontained within an amplification product from a DNA sample isolatedfrom a melon, wherein the amplification product is produced by anamplification reaction using pairs of oligonucleotide primers comprisingthe following nucleotide sequences: SEQ ID NOs: 1 and 2; SEQ ID NOs: 3and 4; SEQ ID NOs: 5 and 6; SEQ ID NOs: 7 and 8; SEQ ID NOs: 9 and 10;SEQ ID NOs: 11 and 12; SEQ ID NOs: 13 and 14; SEQ ID NOs: 15 and 16; SEQID NOs: 17 and 18; SEQ ID NOs: 19 and 20; or SEQ ID NOs: 21 and 22. Insome embodiments, the probe comprises an isolated and purified geneticmarker as disclosed herein and a detectable moiety.

The presently disclosed subject matter also provides improved melonplants, seeds, and tissue cultures produced by any of the presentlydisclosed methods.

The presently disclosed subject matter also provides improved melonplants or a part thereof, which evidences a resistance response to F.oxysporum f.sp. melonis (FOM) race 1,2, comprising a genome homozygouswith respect to one or more genetic alleles which are native to a firstparent and non-native to a second parent of the improved melon plant. Insome embodiments, (a) the second parent evidences less resistanceresponse to FOM race 1,2 than the first parent; and (b) the improvedplant comprises one or more alleles from the first parent that evidenceresistance to FOM race 1,2 in hybrid combination in at least one locusselected from (i) a locus mapping to linkage group 3 and mapped by oneor more of the markers 3.1 and 3.2; (ii) a locus mapping to linkagegroup 6 and mapped by one or more of the markers 6.1 and 6.2; (iii) alocus mapping to linkage group 7 and mapped by one or more of themarkers 7.1 and 7.2; (iv) a locus mapping to linkage group 9 and mappedby one or more of the markers 9.1, 9.2, and 9.3; and (v) a locus mappingto linkage group 10 and mapped by one or so more of the markers 10.1 and10.2; the resistance is not significantly less than that of the firstparent in the same hybrid combination and yield characteristics whichare not significantly different than those of the second parent in thesame hybrid combination. In some embodiments, the improved melon plantscomprise each of (a) a locus mapping to linkage group 3 and mapped byone or more of the markers 3.1 and 3.2; and (b) a locus mapping tolinkage group 9 and mapped by one or more of the markers 9.1, 9.2, and9.3; and have improved resistance to FOM race 1,2 when compared to asubstantially identical melon plant not comprising the loci. In someembodiments, the improved melon plants or parts thereof comprise progenyof a cross between first and second inbred or hybrid lines, wherein oneor more alleles conferring resistance to FOM race 1,2 are present in ahomozygous state in the genome of one or the other or both of the firstand second inbred or hybrid lines, such that the genome of the first andsecond inbreds or hybrids together donate to the improved melon plant orpart thereof a complement of alleles sufficient to confer the resistanceto FOM race 1,2. In some embodiments, the improved melon plants or partsthereof have orange flesh.

The presently disclosed subject matter also provides FOM race1,2-resistant hybrids, or a part thereof, formed with the presentlydisclosed improved melon plants.

The presently disclosed subject matter also provides melon plants, or apart thereof, formed by selling the presently disclosed FOM race1,2-resistant hybrids.

The presently disclosed subject matter also provides melon plants thatare resistant to FOM race 1,2 occurring in melon produced by thepresently disclosed methods. In some embodiments, the melon plants thatare resistant to FOM race 1,2 occurring in melon are hybrid melons.

The presently disclosed subject matter also provides fruit and seedproduced by the presently disclosed melon plants. In some embodiments,the seed the seed comprises a melon line referred to herein as03MFR001795 and corresponding to the seed deposited with NCIMB Ltd.under the terms of the Budapest Treaty on 27 Apr. 2007 as Accession No.41478, or an ancestor or descendent thereof.

The presently disclosed subject matter also provides introgressedCucumis melo plants or germplasm produced by the presently disclosedmethods. In some embodiments, the introgressed Cucumis melo plantsand/or germplasm are homozygous for orange flesh.

The presently disclosed subject matter also provides compositionscomprising amplification primer pairs capable of initiating DNApolymerization by a DNA polymerase on Cucumis melo nucleic acidtemplates to generate Cucumis melo marker amplicons. In someembodiments, the Cucumis melo marker amplicons correspond to Cucumismelo markers 3.1, 3.2, 6.1, 6.2, 7.1, 7.2, 9.1, 9.2, 9.3, 10.1, or 10.2.In some embodiments, the amplification primer pairs comprise one or morenucleotide sequence pairs selected from the group consisting of SEQ IDNOs: 1 and 2; SEQ ID NOs: 3 and 4; SEQ ID NOs: 5 and 6; SEQ ID NOs: 7and 8; SEQ ID NOs: 9 and 10; SEQ ID NOs: 11 and 12; SEQ ID NOs: 13 and14; SEQ ID NOs: 15 and 16; SEQ ID NOs: 17 and 18; SEQ ID NOs: 19 and 20;and SEQ ID NOs: 21 and 22.

The presently disclosed subject matter also provides Cucumis melo plantshaving improved FOM-1,2 resistance associated with the presence of QTL4as defined herein in an homozygous orange flesh genetic background.

The presently disclosed subject matter also provides melon plants thatare resistant to FOM race 1,2, wherein the plant is a plant of thespecies Cucumis melo, and the plant comprises at least one chromosomalregion that confers FOM race 1,2 resistance, and further wherein the atleast one chromosomal region that confers FOM race 1,2 resistance islinked to at least one marker selected from the group consisting ofmarkers 3.1, 3.2, 6.1, 6.2, 7.1, 7.2, 9.1, 9.2, 9.3, 10.1, or 10.2. Insome embodiments, the melon plant is homozygous for a chromosomal regionthat confers FOM race 1,2 resistance linked to marker 9.1, 9.2, or 9.3,or a combination thereof. In some embodiments, the melon plant ishomozygous for orange flesh.

The presently disclosed subject matter also provides parts of the plantsdefined herein. In some embodiments, the plant part is selected from thegroup consisting of pollen, ovule, leaf, embryo, root, root tip, anther,flower, fruit, stem, shoot, seed; scion, rootstock, protoplast, andcallus.

Thus, it is an object of the presently disclosed subject matter toprovide methods for conveying FOM race 1,2 resistance into non-resistantmelon germplasm.

An object of the presently disclosed subject matter having been statedhereinabove, and which is achieved in whole or in part by the presentlydisclosed subject matter, other objects will become evident as thedescription proceeds when taken in connection with the accompanyingdrawings as best described hereinbelow.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NOs: 1 and 2 are the nucleotide sequences of oligonucleotidesthat can be employed together to amplify marker 3.1 associated with QTL1on chromosome 3.

SEQ ID NOs: 3 and 4 are the nucleotide sequences of oligonucleotidesthat can be employed together to amplify marker 3.2 associated with QTL1on chromosome 3.

SEQ ID NOs: 5 and 6 are the nucleotide sequences of oligonucleotidesthat can be employed together to amplify marker 6.1 associated with QTL2on chromosome 6.

SEQ ID NOs: 7 and 8 are the nucleotide sequences of oligonucleotidesthat can be employed together to amplify marker 6.2 associated with QTL2on chromosome 6.

SEQ ID NOs: 9 and 10 are the nucleotide sequences of oligonucleotidesthat can be employed together to amplify marker 7.1 associated with QTL3on chromosome 7.

SEQ ID NOs: 11 and 12 are the nucleotide sequences of oligonucleotidesthat can be employed together to amplify marker 7.2 associated with QTL3on chromosome 7.

SEQ ID NOs: 13 and 14 are the nucleotide sequences of oligonucleotidesthat can be employed together to amplify marker 9.1 associated with QTL4on chromosome 9.

SEQ ID NOs: 15 and 16 are the nucleotide sequences of oligonucleotidesthat can be employed together to amplify marker 9.2 associated with QTL4on chromosome 9.

SEQ ID NOs: 17 and 18 are the nucleotide sequences of oligonucleotidesthat can be employed together to amplify marker 9.3 associated with QTL4on chromosome 9.

SEQ ID NOs: 19 and 20 are the nucleotide sequences of oligonucleotidesthat can be employed together to amplify marker 10.1 associated withQTL5 on chromosome 10.

SEQ ID NOs: 21 and 22 are the nucleotide sequences of oligonucleotidesthat can be employed together to amplify marker 10.2 associated withQTL5 on chromosome 10.

SEQ ID NO: 23 is a 17 nucleotide sequence derived from M13 that can beplaced as a tag (in some embodiments, a fluorescently-labeled tag) 5′ tonucleotide 1 of any of odd-numbered SEQ ID NOs: 1-21 to aid in thedetermination using a sequencer of the size of the amplificationfragment that is produced when oligonucleotides comprising thesesequences are employed to amplify DNA or RNA extracted from a hybrid orinbred Cucumis melo plant or part thereof.

DETAILED DESCRIPTION

The presently disclosed subject matter relates at least in part to theidentification of one or more quantitative trait loci associated withFusarium oxysporum f.sp. melonis (FOM) race 1,2 resistance in Cucumismelo. Thus, provided herein are methods of conveying FOM race 1,2resistance into non-resistant melon germplasm, which employ one or moreof the identified quantitative trait loci in various approaches.

The presently disclosed subject matter also relates at least in part tothe generation of a recombination event through which a linkage of greenflesh and a particular QTL associated with Fusarium oxysporum f.sp.melonis (FOM) race 1,2 resistance in Cucumis melo has been broken. Thus,provided in accordance with the presently disclosed subject matter is aCucumis melo plant having improved FOM race 1,2 resistance in ahomozygous orange flesh genetic background.

I. DEFINITIONS

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the presently disclosed subject matter belongs.Although any methods, devices, and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently disclosed subject matter, representative methods, devices, andmaterials are now described.

Following long-standing patent law convention, the articles “a”, “an”,and “the” refer to “one or more” when used in this application,including in the claims. For example, the phrase “a marker” refers toone or more markers. Similarly, the phrase “at least one”, when employedherein to refer to an entity, refers to, for example, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, or more of thatentity, including but not, limited to whole number values between 1 and100 and greater than 100.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about”. Accordingly, unless indicated to the contrary, thenumerical parameters set forth in this specification and attached claimsare approximations that can vary depending upon the desired propertiessought to be obtained by the presently disclosed subject matter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

As used herein, the term “allele” refers to any of one or morealternative forms of a gene, all of which relate, to at least one traitor characteristic. In a diploid cell, two alleles of a given gene occupycorresponding loci on a pair of homologous chromosomes, although one ofordinary skill in the art understands that the alleles in any particularindividual do not necessarily represent all of the alleles that arepresent in the species. Since the presently disclosed subject matterrelates to QTLs (i.e., genomic regions that can comprise one or moregenes or regulatory sequences), it is in some instances more accurate torefer to a “haplotype” (i.e., an allele of a chromosomal segment)instead of “allele”. However, in such instances, the term “allele”should be understood to comprise the term “haplotype”.

As used herein, the phrase “associated with” refers to a recognizableand/or assayable relationship between two entities. For example, thephrase “associated with resistance” refers to a trait, locus, QTL, gene,marker, phenotype, etc., or the expression thereof, the presence orabsence of which influences an extent or degree of resistance (e.g.,resistance to FOM race 1,2).

As used herein, the term “backcross”, and grammatical variants thereof,refers to a process in which a breeder crosses a hybrid progeny back toone of the parents, for example, a first generation hybrid F1 with oneof the parental genotypes of the F1 hybrid. In some embodiments, abackcross is performed repeatedly, with a progeny individual of onebackcross being itself backcrossed to the same parental genotype.

The term “chromosome” is used herein in its art-recognized meaning ofthe self-replicating genetic structure in the cellular nucleuscontaining the cellular DNA and bearing in its nucleotide sequence thelinear array of genes. The Cucumis melo chromosome numbers disclosedherein refer to those as set forth in Perin et al., 2002, which relatesto a reference nomenclature system adopted by L'institut National de laRecherchë Agronomique (INRA; Paris, France).

As used herein, the terms “cultivar” and “variety” refer to a group ofsimilar plants that by structural or genetic features and/or performancecan be distinguished from other varieties within the same species.

As used herein, the term “gene” refers to a hereditary unit including asequence of DNA that occupies a specific location on a chromosome andthat contains the genetic instruction for a particular characteristicsor trait in an organism.

As used herein, the term “heterozygous” refers to a genetic conditionexisting when different alleles reside at corresponding loci onhomologous chromosomes.

As used herein, the term “homozygous” refers to a genetic conditionexisting when identical alleles reside at corresponding loci onhomologous chromosomes.

As used herein, the term “hybrid” in the context of nucleic acids refersto a double-stranded nucleic acid molecule, or duplex, formed byhydrogen bonding between complementary nucleotide bases. The terms“hybridize” or “anneal” refer to the process by which single strands ofnucleic acid sequences form double-helical segments through hydrogenbonding between complementary bases.

As used herein, the term “hybrid” in the context of plant breedingrefers to a plant that is the offspring of genetically dissimilarparents produced by crossing plants of different lines or breeds orspecies, including but not limited to the cross between two inbredlines.

As used herein, the term “inbred” refers to a substantially homozygousindividual or line.

As used herein, the terms “introgression”, “introgressed”, and“introgressing” refer to both a natural and artificial process wherebygenomic regions of one species, variety, or cultivar are moved into thegenome of another species, variety, or cultivar, by crossing thosespecies. The process can optionally be completed by backcrossing to therecurrent parent.

As used herein, the term “linkage” refers to a phenomenon whereinalleles on the same chromosome tend to be transmitted together moreoften than expected by chance if their transmission was independent.Thus, two alleles on the same chromosome are said to be “linked” whenthey segregate from each other in the next generation in someembodiments less than 50% of the time, in some embodiments less than 25%of the time, in some embodiments less than 20% of the time, in someembodiments less than 15% of the time, in some embodiments less than 10%of the time, in some embodiments less than 9% of the time, in someembodiments less than 8% of the time, in some embodiments less than 7%of the time, in some embodiments less than 6% of the time, in someembodiments less than 5% of the time, in some embodiments less than 4%of the time, in some embodiments less than 3% of the time, in someembodiments less than 2% of the time, and in some embodiments less than1% of the time.

In some embodiments, “linkage” implies physical proximity on achromosome. Thus, two loci are linked if they are within in someembodiments 20, in some embodiments 15, in some embodiments 12, in someembodiments 10, in some embodiments 9, in some embodiments 8, in someembodiments 7, in some embodiments 6, in some embodiments 5, in someembodiments 4, in some embodiments 3, in some embodiments 2, and in someembodiments 1 centiMorgans (cM) of each other. Similarly, a QTL islinked to a marker if it is in some embodiments within 20, 15, 12, 10,9, 8, 7, 6, 5, 4, 3, 2, or 1 cM of the marker.

As used herein, the phrase “linkage group” refers to all of the genes orgenetic traits that are located on the same chromosome. Within thelinkage group, those loci that are close enough together can exhibitlinkage in genetic crosses. Since the probability of crossover increaseswith the physical distance between loci on a chromosome, loci for whichthe locations are far removed from each other within a linkage groupmight not exhibit any detectable linkage in direct genetic tests. Theterm “linkage group” is mostly used to refer to genetic loci thatexhibit linked behavior in genetic systems where chromosomal assignmentshave not yet been made. Thus, in the present context, the term “linkagegroup” is synonymous with the physical entity of a chromosome, althoughone of ordinary skill in the art will understand that a linkage groupcan also be defined as corresponding to a region of (i.e., less than theentirety) of a given chromosome.

As used herein, the term “locus” refers to a position that a given geneor a regulatory sequence occupies on a chromosome of a given species.

As used herein, the term “marker” refers to an identifiable position ona chromosome the inheritance of which can be monitored. In someembodiments, a marker comprises a known or detectable nucleic acidsequence.

In some embodiments, a marker corresponds to an amplification productgenerated by amplifying a Cucumis melo nucleic acid with twooligonucleotide primers, for example, by the polymerase chain reaction(PCR). As used herein, the phrase “corresponds to an amplificationproduct” in the context of a marker refers to a marker that has anucleotide sequence that is the same (allowing for mutations introducedby the amplification reaction itself) as an amplification product thatis generated by amplifying Cucumis melo genomic DNA with a particularset of primers. In some embodiments, the amplifying is by PCR, and theprimers are PCR primers that are designed to hybridize to oppositestrands of the Cucumis melo genomic DNA in order to amplify a Cucumismelo genomic DNA sequence present between the sequences to which the PCRprimers hybridize in the Cucumis melo genomic DNA. The amplifiedfragment that results from one or more rounds of amplification usingsuch an arrangement of primers is a double stranded nucleic acid, onestrand of which has a nucleotide sequence that comprises, in 5′ to 3′order, the sequence of one of the primers', the sequence of the Cucumismelo genomic DNA located between the primers, and the reverse-complementof the second primer. Typically, the “forward” primer is assigned to bethe primer that has the same sequence as a subsequence of the(arbitrarily assigned) “top” strand of a double-stranded nucleic acid tobe amplified, such that the “top” strand of the amplified fragmentincludes a nucleotide sequence that is, in 5′ to 3′ direction, equal tothe sequence of the forward primer—the sequence located between theforward and reverse primers of the top strand, of the genomicfragment—the reverse-complement of the reverse primer. Accordingly, amarker that “corresponds to” an amplified fragment is a marker that hasthe same sequence of one of the strands of the amplified fragment.

As used herein, the term “melon” refers to a plant, or a part thereof,of the species Cucumis melo, also referred to herein as Cucumis melo L.

As used herein, the phrase “melon-specific DNA sequence” refers to apolynucleotide sequence having a nucleotide sequence homology of in someembodiments more than 50%, in some embodiments more than 55%, in someembodiments more than 60%, in some embodiments more than 65%, in someembodiments more than 70%, in some embodiments more than 75%, in someembodiments more than 80%, in some embodiments more than 85%, in someembodiments more than 90%, in some embodiments more than 92%, in someembodiments more than 95%, in some embodiments more than 96%, in someembodiments more than 97%, in some embodiments more than 98%, and insome embodiments more than 99% with a sequence of the genome of thespecies Cucumis melo that shows the greatest similarity to it, in someembodiments in the case of markers for any of QTL1-QTL5, the part of theDNA sequence of a melon flanking the QTL1-QTL5 markers.

As used herein, the phrase “molecular marker” refers to an indicatorthat is used in methods for visualizing differences in characteristicsof nucleic acid sequences. Examples of such indicators are restrictionfragment length polymorphism (RFLP) markers, amplified fragment lengthpolymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs),insertion mutations, microsatellite markers (SSRs),sequence-characterized amplified regions (SCARS), cleaved amplifiedpolymorphic sequence (CAPS) markers or isozyme markers or combinationsof the markers described herein which defines a specific genetic andchromosomal location. A “molecular marker linked to a QTL” as definedherein can thus refer to SNPs, insertion mutations, as well as moreusual AFLP markers or any other type of marker used in the field.

As used herein, the phrase “nucleotide sequence homology” refers to thepresence of homology between two polynucleotides. Polynucleotides have“homologous” sequences if the sequence of nucleotides in the twosequences is the same when aligned for maximum correspondence. Sequencecomparison between two or more polynucleotides is generally performed bycomparing portions of the two sequences over a comparison window toidentify and compare local regions of sequence similarity. Thecomparison window is generally from about 20 to 200 contiguousnucleotides. The “percentage of sequence homology” for polynucleotides,such as 50, 60, 70, 80, 90, 95, 98, 99 or 100 percent sequence homologycan be determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may include additions or deletions (i.e., gaps) ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The percentage iscalculated by: (a) determining the number of positions at which theidentical nucleic acid base occurs in both sequences to yield the numberof matched positions; (b) dividing the number of matched positions bythe total number of positions in the window of comparison; and (c)multiplying the result by 100 to yield the percentage of sequencehomology. Optimal alignment of sequences for comparison may be conductedby computerized implementations of known algorithms, or by visualinspection. Readily available sequence comparison and multiple sequencealignment algorithms are, respectively, the Basic Local Alignment SearchTool (BLAST; Altschul et al., 1990; Altschul et al., 1997) and ClustalWprograms, both available on the internet. Other suitable programsinclude, but are not limited to, GAP, BestFit, PlotSimilarity, andFASTA, which are part of the Accelrys GCG Package available fromAccelrys, Inc. of San Diego, Calif., United States of America.

As used herein, the term “offspring” plant refers to any plant resultingas progeny from a vegetative or sexual reproduction from one or moreparent plants or descendants thereof. For instance an offspring plantcan be obtained by cloning or setting of a parent plant or by crossingtwo parent plants and include selfings as well as the F1 or F2 or stillfurther generations. An F1 is a first-generation offspring produced fromparents at least one of which is used for the first time as donor of atrait, while offspring of second generation (F2) or subsequentgenerations (F3, F4, and the like.) are specimens produced from selfingsof F1s, F2s and the like. An F1 can thus be (and in some embodiments is)a hybrid resulting from a cross between two true breeding parents(true-breeding is homozygous for a trait), while an F2 can be (and insome embodiments is) an offspring resulting from self-pollination of theF1 hybrids.

As used herein, the term “phenotype” refers to a detectablecharacteristic of a cell or organism, which characteristics are at leastpartially a manifestation of gene expression.

As used herein, the phrase “plant part” refers to a part of a plant,including single cells and cell tissues such as plant cells that areintact in plants, cell clumps, and tissue cultures from which plants canbe regenerated. Examples of plant parts include, but are not limited to,single cells and tissues from pollen, ovules, leaves, embryos, roots,root tips, anthers, flowers, fruits, sterns, shoots, and seeds; as wellas scions, rootstocks, protoplasts, calli, and the like.

As used herein, the term “population” refers to a geneticallyheterogeneous collection of plants sharing a common genetic derivation.

As used herein, the term “primer” refers to an oligonucleotide which iscapable of annealing to a nucleic acid target allowing a DNA polymeraseto attach, thereby serving as a point of initiation of DNA synthesiswhen placed under conditions in which synthesis of a primer extensionproduct is induced (e.g., in the presence of nucleotides and an agentfor polymerization such as DNA polymerase and at a suitable temperatureand pH).

The primer (in some embodiments an extension primer and in someembodiments an amplification primer) is in some embodiments singlestranded for maximum efficiency in extension and/or amplification. Insome embodiments, the primer is an oligodeoxyribonucleotide.

A primer is typically sufficiently long to prime the synthesis ofextension and/or amplification products in the presence of the agent forpolymerization. The minimum lengths of the primers can depend on manyfactors, including, but not limited to temperature and composition (A/Tvs. G/C content) of the primer.

In the context of an amplification primer, these are typically providedas a pair of bi-directional primers consisting of one forward and onereverse primer as commonly used in the art of DNA amplification such asin PCR amplification.

As such, it will be understood that the term “primer”, as used herein,can refer to more than one primer, particularly in the case where thereis some ambiguity in the information regarding the terminal sequence(s)of the target region to be amplified. Hence, a “primer” can include acollection of primer oligonucleotides containing sequences representingthe possible variations in the sequence or includes nucleotides whichallow a typical base pairing.

Primers can be prepared by any suitable method. Methods for preparingoligonucleotides of specific sequence are known in the art, and include,for example, cloning and restriction of appropriate sequences and directchemical synthesis. Chemical synthesis methods can include, for example,the phospho di- or tri-ester method, the diethylphosphoramidate methodand the solid support method disclosed in U.S. Pat. No. 4,458,066.

Primers can be labeled, if desired, by incorporating detectable moietiesby for instance spectroscopic, fluorescence, photochemical, biochemical,immunochemical, or chemical moieties.

Template-dependent extension of an oligonucleotide primer is catalyzedby a polymerizing agent in the presence of adequate amounts of the fourdeoxyribonucleotide triphosphates (dATP, dGTP, dCTP and dTTP; i.e.,dNTPs) or analogues, in a reaction medium that comprises appropriatesalts, metal cations, and a pH buffering system. Suitable polymerizingagents are enzymes known to catalyze primer- and template-dependent DNAsynthesis. Known DNA polymerases include, for example, E. coli DNApolymerase I or its Klenow fragment, T4 DNA polymerase, and Taq DNApolymerase, as well as various modified versions thereof. The reactionconditions for catalyzing DNA synthesis with these DNA polymerases areknown in the art. The products of the synthesis are duplex moleculesconsisting of the template strands and the primer extension strands,which include the target sequence. These products, in turn, can serve astemplate for another round of replication. In the second round ofreplication, the primer extension strand of the first cycle is annealedwith its complementary primer; synthesis yields a “short” product whichis bound on both the 5′- and the 3′-ends by primer sequences or theircomplements. Repeated cycles of denaturation, primer annealing, andextension result in the exponential accumulation of the target regiondefined by the primers. Sufficient cycles are run to achieve the desiredamount of polynucleotide containing the target region of nucleic acid.The desired amount can vary, and is determined by the function which theproduct polynucleotide is to serve.

The PCR method is well described in handbooks and known to the skilledperson. After amplification by PCR, the target polynucleotides can bedetected by hybridization with a probe polynucleotide which forms astable hybrid with that of the target sequence under stringent tomoderately stringent hybridization and wash conditions. If it isexpected that the probes will be essentially completely complementary(i.e., about 99% or greater) to the target sequence, stringentconditions can be used. If some mismatching is expected, for example ifvariant strains are expected with the result that the probe will not becompletely complementary, the stringency of hybridization can bereduced. In some embodiments, conditions are chosen to rule outnon-specific/adventitious binding. Conditions that affect hybridization,and that select against non-specific binding are known in the art, andare described in, for example, Sambrook & Russell, 2001. Generally,lower salt concentration and higher temperature increase the stringencyof hybridization conditions.

Continuing, the term “probe” refers to a single-stranded oligonucleotidesequence that will form a hydrogen-bonded duplex with a complementarysequence in a target nucleic acid sequence analyte or its cDNAderivative.

As used herein, the terms “QTL1”, “QTL2”, “QTL3”, “QTL4”, and “QTL5”refer to the genomic regions linked to FOM race 1,2 resistance asdefined by the markers 3.1 and 3.2; 6.1 and 6.2; 7.1 and 7.2; 9.1, 9.2,and 9.3; and 10.1 and 10.2; respectively. For the purposes of theinstant disclosure, these markers are said to be present on Cucumis melochromosomes 3, 6, 7, 9, and 10, respectively.

As used herein, the term “quantitative trait locus” (QTL; pluralquantitative trait loci; QTLs) refers to a genetic locus (or loci) thatcontrol to some degree a numerically representable trait that, in someembodiments, is continuously distributed. As such, the term QTL is usedherein in its art-recognized meaning to refer to a chromosomal regioncontaining alleles (e.g., in the form of genes or regulatory sequences)associated with the expression of a quantitative phenotypic trait. Thus,a QTL “associated with” resistance to FOM race 1,2 refers to one or moreregions located on one or more chromosomes that includes at least onegene the expression of which influences a level of resistance and/or atleast one regulatory region that controls the expression of one or moregenes involved in resistance to FOM race 1,2. The QTLs can be defined byindicating their genetic location in the genome of a specific Cucumismelo accession using one or more molecular genomic markers. One or moremarkers, in turn, indicate a specific locus. Distances between loci areusually measured by the frequency of crossovers between loci on the samechromosome. The farther apart two loci are, the more likely that acrossover will occur between them. Conversely, if two loci are closetogether, a crossover is less likely to occur between them. Typically,one centiMorgan 1.5 (CM) is equal to 1% recombination between loci. Whena QTL can be indicated by multiple markers, the genetic distance betweenthe end-point markers is indicative of the size of the QTL.

As used herein, the term “recombination” refers to an exchange (a“crossover”) of DNA fragments between two DNA molecules or chromatids ofpaired chromosomes over in a region of similar or identical nucleotidesequences. A “recombination event” is herein understood to refer to ameiotic crossover.

As used herein, the term “regenerate”, and grammatical variants thereof,refers to the production of a plant from tissue culture.

As used herein, the term “resistant” and “resistance” encompass bothpartial and full resistance to infection (e.g., infection by FOM race1,2). A susceptible plant can either be non-resistant or have lowerlevels of resistance to infection relative to a resistant plant. Theterm is used to include such separately identifiable forms of resistanceas “full resistance”, “immunity”, “intermediate resistance”, “partialresistance”, “hypersensitivity”, and “tolerance”.

As used herein, the phrase “stringent hybridization conditions” refersto conditions under which a polynucleotide hybridizes to its targetsubsequence, typically in a complex mixture of nucleic acids, but toessentially no other sequences. Stringent conditions aresequence-dependent and can be different under different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, 1993. Generally, stringent conditions are selected to be about5-10° C. lower than the thermal melting point (Tm) for the specificsequence at a defined ionic strength pH. The Tm is the temperature(under defined ionic strength, pH, and nucleic acid concentration) atwhich 50% of the probes complementary to the target hybridize to thetarget sequence at equilibrium (as the target sequences are present inexcess, at Tm, 50% of the probes are occupied at equilibrium). Stringentconditions are those in which the salt concentration is less than about1:0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration(or other salts) at pH 7.0 to 8.3 and the temperature is at least about30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about60° C. for long probes (e.g., greater than 50 nucleotides). Stringentconditions can also be achieved with the addition of destabilizingagents such as formamide. For selective or specific hybridization, apositive signal is in some embodiments at least two times background,and in some embodiments 10 times background hybridization. Exemplarystringent hybridization conditions include: 50% formamide, 5×SSC, and 1%SDS, incubating at 42° C.; or 5×SSC, 1% SOS, incubating at 65° C.; withone or more washes in 0.2×SSC and 0.1% SDS at 65° C. For PCR, atemperature of about 36° C. is typical for low stringency amplification,although annealing temperatures can vary between about 32° C. and 48° C.(or higher) depending on primer length. Additional guidelines fordetermining hybridization parameters are provided in numerous references(see e.g., Ausubel et al., 1999).

As used herein, the term “susceptible” refers to a plant having noresistance to the disease resulting in the plant being affected by thedisease, resulting in disease symptoms. The term “susceptible” istherefore equivalent to “non-resistant”. Alternatively, the term“susceptible” can be employed in a relative context, in which one plantis considered “susceptible” because it is less resistant to a particularpathogen than is a second plant (which in the context of these terms ina relative usage, would be referred to as the “resistant” plant”).

II. PLANT BREEDING

The purpose of breeding programs in agriculture and horticulture is toenhance the performances of plants by improving their geneticcomposition. In essence, this improvement accrues by increasing thefrequency of the most favorable alleles for the genes influencing theperformance characteristics of interest. Wild plant lines provide a richresource of genetic and phenotypic variation. Traditionally,agricultural or horticultural practice makes use of this variation byselecting a wild plant line or its offspring for having desiredgenotypic or potential phenotypic properties, crossing it with a linehaving additional desired genotypic or potential phenotypic propertiesand selecting from among the offspring plants those that exhibit thedesired genotypic or potential phenotypic properties (or an increasedfrequency thereof).

A growing understanding and utilization of the laws of Mendelianinheritance in combination with molecular genetic tools have in the pastcentury facilitated this selection process. For example, methods forselecting plants for having desired genotypic or potential phenotypicproperties have become available based on testing the plant for thepresence of a quantitative trait locus (QTL); i.e., for the presence ofa chromosomal region containing alleles associated with the expressionof a continuously distributed (quantitative) phenotypic trait. Usually aQTL is characterized by one or more markers that statistically associateto the quantitative variation in the phenotypic trait and is essentiallysynonymous to a gene. QTL mapping allows for the identification ofcandidate loci affecting the expression of a trait of interest. In plantbreeding, it allows for marker-assisted selection (MAS); i.e., theselection of plants having favorable alleles by detecting in thoseplants the QTL-associated markers.

One of the major problems in breeding programs of cultivated plants isthe existence of negative genetic correlation between separate traits.This is for example the case with the negative genetic correlationbetween reproductive capacity and production in variousdisease-resistant plant lines. Understanding emerges to show thatintrogressions of DNA from the genome of one plant line into another caninterfere with and/or otherwise negatively affect the expression ofbasic reproductive traits. Likewise, attempts to introgressresistance-conferring gene sequences from one plant into another canremove resistance traits already present in the recipient line.

Knowledge of the inheritance of various traits allows for the selectionof lines homozygous for a QTL associated with disease resistance. Use ofthe knowledge of the genetic origin and location of a desired trait in abreeding program can increase the accuracy of the predicted breedingoutcome and can enhance the rate of selection compared to conventionalbreeding programs. For instance, the fact that the genetic basis of adesired trait is heritably linked to another trait can help to increaseuniformity for those two traits among the offspring since a parenthomozygous for the desired alleles will pass them to most if not alloffspring, resulting in a reduced segregation in the offspring.

The presently disclosed subject matter provides for better models formarker-assisted selection (MAS). The presently disclosed subject mattertherefore relates to methods of plant breeding and to methods to selectplants, in particular melon plants, particularly cultivated melon plantsas breeder plants for use in breeding programs or cultivated melonplants for having desired genotypic or potential phenotypic properties,in particular related to producing valuable melons, also referred toherein as commercially valuable plants. Herein, a cultivated plant isdefined as a plant being purposely selected or having been derived froma plant having been purposely selected in agricultural or horticulturalpractice for having desired genotypic or potential phenotypicproperties, for example a plant obtained by inbreeding.

The presently disclosed subject matter thus also provides methods forselecting a plant of the species. Cucumis melo exhibiting resistancetowards FOM race 1,2 comprising detecting in the plant the presence ofone or more of QTL1-QTL5 as defined herein. In an exemplary embodimentof the presently disclosed methods for selecting such a plant, themethod comprises providing a sample of genomic DNA from a melon plant;and (b) detecting in the sample of genomic DNA at least one molecularmarker linked to a QTL selected from the group consisting of QTL1-QTL5.In some embodiments, the detecting can comprise detecting at least twomolecular markers from the group, the at least two molecular markersdetecting at least two (e.g., 2, 3, 4, or 5) of QTL1-QTL5.

The providing of a sample of genomic DNA from a melon plant can beperformed by standard DNA isolation methods well known in the art.

The detecting of a molecular marker can in some embodiments comprise theuse of one or more sets of primer pairs that can be used to produce oneor more amplification products that are suitable markers for one of theQTLs. Such a set of primers can comprise, in some embodiments,nucleotide sequences as set forth in SEQ ID NOs: 1-22.

In some embodiments, the detecting of a molecular marker (step b) cancomprise the use of a nucleic acid probe having a base sequence that issubstantially complementary to the nucleic acid sequence defining themolecular marker and which nucleic acid probe specifically hybridizesunder stringent conditions with a nucleic acid sequence defining themolecular marker. A suitable nucleic acid probe can for instance be asingle strand of the amplification product corresponding to the marker.

The detecting of a molecular marker can also comprise the performance ofa nucleic acid amplification reaction on the genomic DNA to detect oneor more QTLs. This can be done by performing a PCR reaction using a setof marker-specific primers. In some embodiments, the detecting cancomprise the use of at least one set of primers defining one or moremarkers linked to one or more of QTL1-QTL5, or a set of primers whichspecifically hybridize under stringent conditions with nucleic acidsequences of one or more markers linked to one or more of QTL1-QTL5.

The presently disclosed methods can also include detecting an amplifiedDNA fragment associated with the presence of a QTL. In some embodiments,the amplified fragment associated with presence of a QTL has a predictedlength or nucleic acid sequence, and detecting an amplified DNA fragmenthaving the predicted length or the predicted nucleic acid sequence isperformed such that the amplified DNA fragment has a length thatcorresponds (plus or minus a few bases; e.g., a length of one, two orthree bases more or less) to the expected length as based on a similarreaction with the same primers with the DNA from the plant in which themarker was first detected or the nucleic acid sequence that corresponds(has a homology of in some embodiments more than 80%, in someembodiments more than 90%, in some embodiments more than 95%, in someembodiments more than 97%, and in some embodiments more than 99%) to theexpected sequence as based on the sequence of the marker associated withthat QTL in the plant in which the marker was first detected. Upon areview of the instant disclosure, one of ordinary skill in the art wouldappreciate that markers that are absent in resistant plants, while theywere present in the susceptible parent(s) (so-called trans-markers), canalso be useful in assays for detecting resistance among offspringplants, although testing the absence of a marker to detect the presenceof a specific trait is not optimal.

The detecting of an amplified DNA fragment having the predicted lengthor the predicted nucleic acid sequence can be performed by any of anumber or techniques, including but not limited to standardgel-electrophoresis techniques or by using automated DNA sequencers. Themethods are not described here in detail as they are well known to theskilled person, although exemplary approaches are set forth in EXAMPLES5 and 6.

In order to detect in a plant the presence of two QTLs on a singlechromosome, chromosome painting methods can also be used. In suchmethods at least a first QTL and at least a second QTL can be detectedin the same chromosome by in situ hybridization or in situ PCRtechniques. More conveniently, the fact that two QTLs are present on asingle chromosome can be confirmed by determining that they are incoupling phase; i.e., that the traits show reduced segregation whencompared to genes residing on separate chromosomes.

III. MOLECULAR MARKERS AND QTLs

Molecular markers are used for the visualization of differences innucleic acid sequences. This visualization can be due to DNA-DNAhybridization techniques after digestion with a restriction enzyme(RFLP) and/or due to techniques using the polymerase chain reaction(e.g., STS, SSR/microsatellites, AFLP, and the like.). In someembodiments, all differences between two parental genotypes segregate ina mapping population based on the cross of these parental genotypes. Thesegregation of the different markers can be compared and recombinationfrequencies can be calculated. Methods for mapping markers in plants aredisclosed in, for example, Glick & Thompson, 1993; Zietkiewicz et al.,1994.

The recombination frequencies of molecular markers on differentchromosomes are generally 50%. Between molecular markers located on thesame chromosome, the recombination frequency generally depends on thedistance between the markers. A low recombination frequency correspondsto a low genetic distance between markers on a chromosome. Comparing allrecombination frequencies results in the most logical order of themolecular markers on the chromosomes. This most logical order can bedepicted in a linkage map (Paterson, 1996). A group, of adjacent orcontiguous markers on the linkage map that is associated with anincreased level of resistance to a disease; e.g., to a reduced incidenceof acquiring the disease upon infectious contact with the disease agentand/or a reduced lesion growth rate upon establishment of infection, canprovide the position of a QTL associated with resistance to thatdisease.

The markers identified herein can be used is various aspects of thepresently disclosed subject matter as set forth hereinbelow. Aspects ofthe presently disclosed subject matter are not to be limited to the useof the markers identified herein, however. It is stressed that theaspects can also make use of markers not explicitly disclosed herein oreven yet to be identified. Other than the genetic unit “gene”, on whichthe phenotypic expression depends on a large number of factors thatcannot be predicted, the genetic unit “QTL” denotes a region on thegenome that is directly related to a phenotypic quantifiable trait.

The five QTLs identified herein are located on five differentchromosomes or linkage groups and their locations can be characterizedby a number of otherwise arbitrary markers. In the presentinvestigations, microsatellite markers (e.g., SSRs), were used, althoughrestriction fragment length polymorphism (RFLP) markers, amplifiedfragment length polymorphism (AFLP) markers, single nucleotidepolymorphisms (SNPs), insertion mutation markers, sequence-characterizedamplified region (SCAR) markers, cleaved amplified polymorphic sequence(CAPS) markers or isozyme markers or combinations of these markers mightalso have been used, and indeed can be used.

In general, a QTL can span a region of several million bases. Therefore,providing the complete sequence information for the QTL is practicallyunfeasible but also unnecessary, as the way in which the QTL is firstdetected—through the observed correlation between the presence of astring of contiguous genomic markers and the presence of a particularphenotypic trait—allows one to trace among a population of offspringplants those plants that have the genetic potential for exhibiting aparticular phenotypic trait. By providing a non-limiting list ofmarkers, the presently disclosed subject matter thus provides for theeffective use of the presently disclosed QTLs in a breeding program.

In some embodiments, a marker is specific for a particular line ofdescent. Thus, a specific trait can be associated with a particularmarker. The markers as disclosed herein not only indicate the locationof the QTL, they also correlate with the presence of the specificphenotypic trait in a plant. It is noted that the contiguous genomicmarkers that indicate the location of the QTL on the genome are inprincipal arbitrary or non-limiting. In general, the location of a QTLis indicated by a contiguous string of markers that exhibit statisticalcorrelation to the phenotypic trait. Once a marker is found outside thatstring (i.e., one that has a LOD-score below a certain threshold,indicating that the marker is so remote that recombination in the regionbetween that marker and the QTL occurs so frequently that the presenceof the marker does not correlate in a statistically significant mannerto the presence of the phenotype) the boundaries of the QTL can beconsidered set. Thus, it is also possible to indicate the location ofthe QTL by other markers located within that specified region.

It is further noted that the contiguous genomic markers can also be usedto indicate the presence of the QTL (and thus of the phenotype) in anindividual plant, which in some embodiments means that they can be usedin marker-assisted selection (MAS) procedures. In principle, the numberof potentially useful markers is limited but can be very large, and oneof ordinary skill in the art can easily identify markers in addition tothose specifically disclosed in the present application. Any marker thatis linked to the QTL (e.g., falling within the physically boundaries ofthe genomic region spanned by the markers having established LOD scoresabove a certain threshold thereby indicating that no or very littlerecombination between the marker and the QTL occurs in crosses, as wellas any marker in linkage disequilibrium to the QTL, as well as markersthat represent the actual causal mutations within the QTL) can be usedin MAS procedures. This means that the markers identified in theapplication as associated to the QTLs, such as the SSR markers 3.1, 3.2,6.1, 6.2, 7.1, 7.2, 9.1, 9.2, 9.3, 10.1, and 10.2 QTL1-QTL5, are mereexamples of markers suitable for use in MAS procedures. Moreover, whenthe QTL, or the specific trait-conferring part thereof, is introgressedinto another genetic background (i.e., into the genome of another melonor another plant species), then some markers might no longer be found inthe offspring although the trait is present therein, indicating thatsuch markers are outside the genomic region that represents the specifictrait-conferring part of the QTL in the original parent line only andthat the new genetic background has a different genomic organization.Such markers of which the absence indicates the successful introductionof the genetic element in the offspring are called “trans markers” andcan be equally suitable in MAS procedures under the presently disclosedsubject matter.

Upon the identification of a QTL, the QTL effect (e.g., the resistance)can for instance be confirmed by assessing resistance in progenysegregating for the QTLs under investigation. The assessment of theresistance can suitably be performed by using a resistance bioassay asknown in the art for FOM race 1,2. For example, (field) trials undernatural and/or artificial infection conditions can be conducted toassess the resistance of hybrid and/or inbred melons to FOM race 1,2, orif desired, any other FOM race.

The markers provided by the presently disclosed subject matter can beused for detecting the presence of one or more FOM race 1,2 resistancealleles at QTLs of the presently disclosed subject matter in a suspectedFOM-resistant melon plant, and can therefore be used in methodsinvolving marker-assisted breeding and selection of FOM (e.g., FOM race1,2) resistant melon plants. In some embodiments, detecting the presenceof a QTL of the presently disclosed subject matter is performed with atleast one of the markers for a QTL as defined herein. The presentlydisclosed subject matter therefore relates in another aspect to a methodfor detecting the presence of a QTL for FOM race 1,2 resistance,comprising detecting the presence of a nucleic acid sequence of the QTLin a suspected FOM race 1,2-resistant melon plant, which presence can bedetected by the use of the disclosed markers.

The nucleotide sequence of a QTL of the presently disclosed subjectmatter can for instance be resolved by determining the nucleotidesequence of one or more markers associated with the QTL and designinginternal primers for the marker sequences that can then be used tofurther determine the sequence of the QTL outside of the markersequences. For instance, the nucleotide sequence of the SSR markersdisclosed herein can be obtained by isolating the markers from theelectrophoresis gel used in the determination of the presence of themarkers in the genome of a subject plant, and determining the nucleotidesequence of the markers by, for example, dideoxy chain terminationsequencing methods, which are well known in the art.

In embodiments of such methods for detecting the presence of a QTL in asuspected FOM race 1,2-resistant melon plant, the method can alsocomprise providing a oligonucleotide or polynucleotide capable ofhybridizing under stringent hybridization conditions to a nucleic acidsequence of a marker linked to the QTL, in some embodiments selectedfrom the markers disclosed herein, contacting the oligonucleotide orpolynucleotide with digested genomic nucleic acid of a suspected FOMrace 1,2-resistant melon plant, and determining the presence of specifichybridization of the oligonucleotide or polynucleotide to the digestedgenomic nucleic acid.

In some embodiments, the method is performed on a nucleic acid sampleobtained from the suspected FOM race 1,2-resistant melon plant, althoughin situ hybridization methods can also be employed. Alternatively, oneof ordinary skill in the art can, once the nucleotide sequence of theQTL has been determined, design specific hybridization probes oroligonucleotides capable of hybridizing under stringent hybridizationconditions to the nucleic acid sequence of the QTL and can use suchhybridization probes in methods for detecting the presence of a QTLdisclosed herein in a suspected FOM race 1,2-resistant melon plant.

IV. PRODUCTION OF FOM RACE 1,2-RESISTANT MELON PLANTS BY TRANSGENICMETHODS

According to another aspect of the presently disclosed subject matter, anucleic acid (in some embodiments a DNA) sequence comprising, one ormore of QTL1-QTL5 or resistance-conferring parts thereof, can be usedfor the production of a resistant melon plant of the presently disclosedsubject matter. In this aspect, the presently disclosed subject matterprovides for the use of QTLs as defined herein or resistance-conferringparts thereof, for producing a resistant melon plant, which use involvesthe introduction of a nucleic acid sequence comprising the QTL into asuitable recipient plant. As stated, the nucleic acid sequence can bederived from a suitable FOM race 1,2-resistant donor plant. A suitablesource for the FOM race 1,2 resistance locus identified herein as any ofQTL1-QTL5 is Rupia, originating from Japan (Mikado Seed Growers Co.Ltd., Chiba City, Japan).

A number of melon cultivars that have varying degrees of resistance toFOM race 1,2 are commercially available. Melon plants that havedemonstrated some resistance to FOM race 1,2 resistant include, but arenot limited to Isabelle (INRA); Manta, Tolosa, Targa, Tadeo, and Flavio(Clause Tezier SA, Valence, France); Sting (Nunhems, Netherlands BV,Haelen, The Netherlands); and Raffal, Zeffir, Helfi, Neffiac, and Fidji(Gautier Graines SA, Eyragues, France).

The source of the resistance loci described herein is the Cucumis meloL. cv. Rupia F1 cultivar (Mikado Seed Growers Co. Ltd.), which wasoriginally generated by crossing an orange-fleshed Japanese Cucumis melowith an unknown resistant melon that was green-fleshed. However, theRupia F1 is thus heterozygous for flesh color, which is undesirable forthe purposes of commercial melon production.

Additionally, it was determined through the experiments disclosed hereinthat at least one of the FOM race 1,2 resistance alleles present withinthe genome of Rupia was very tightly linked to a green flesh colorallele within QTL4 present on chromosome 9. For various reasons, it isdesirable to produce a Cucumis melo variety that is (a) homozygous fororange flesh (i.e., lacks green flesh alleles); and (b) Includes the FOMrace 1,2 resistance alleles present on chromosome 9. In order toaccomplish this, the chromosome 9 green flesh allele(s) and FOM race 1,2resistance allele(s) had to be segregated from each other in a progenyplant derived from breeding Rupia (or a descendent thereof) to anotherCucumis melo line (e.g., an orange flesh color line). This has now beenaccomplished, as disclosed herein.

Once identified in a suitable donor plant, the nucleic acid sequencethat comprises a QTL for FOM race 1,2-resistance, or aresistance-conferring part thereof, can be transferred to a suitablerecipient plant by any method available. For instance, the nucleic acidsequence can be transferred by crossing a FOM race 1,2-resistant donorplant with a susceptible recipient plant (i.e., by introgression), bytransformation, by protoplast fusion, by a doubled haploid technique, byembryo rescue, or by any other nucleic acid transfer system, optionallyfollowed by selection of offspring plants comprising one or more of thepresently disclosed QTLs and exhibiting resistance. For transgenicmethods of transfer, a nucleic, acid sequence comprising a QTL for FOMrace 1,2-resistance, or a resistance-conferring part thereof, can beisolated from the donor plant using methods known in the art, and thethus isolated nucleic acid sequence can be transferred to the recipientplant by transgenic methods, for instance by means of a vector, in agamete, or in any other suitable transfer element, such as a ballisticparticle coated with the nucleic acid sequence.

Plant transformation generally involves the construction of anexpression vector that will function in plant cells. In the presentlydisclosed subject matter, such a vector comprises a nucleic acidsequence that comprises a QTL for FOM race 1,2 resistance, or aresistance-conferring part thereof, which vector can comprise a FOM race1,2-conferring gene that is under control of or operatively linked to aregulatory element, such as a promoter. The expression vector cancontain one or more such operably linked gene/regulatory elementcombinations, provided that at least one of the genes contained in thecombinations encodes FOM race 1,2-resistance. The vector(s) can be inthe form of a plasmid, and can be used, alone or in combination withother plasmids, to provide transgenic plants that are resistant to FOMrace 1,2, using transformation methods known in the art, such as theAgrobacterium transformation system.

Expression vectors can include at least one marker gene, operably linkedto a regulatory element (such as a promoter) that allows transformedcells containing the marker to be either recovered by negative selection(by inhibiting the growth of cells that do not contain the selectablemarker gene), or by positive selection (by screening for the productencoded by the marker gene). Many commonly used selectable marker genesfor plant transformation are known in the art, and include, for example,genes that code for enzymes that metabolically detoxify a selectivechemical agent that can be an antibiotic or a herbicide, or genes thatencode an altered target which is insensitive to the inhibitor. Severalpositive selection methods are known in the art, such as mannoseselection. Alternatively, marker-less transformation can be used toobtain plants without the aforementioned marker genes, the techniquesfor which are also known in the art.

One method for introducing an expression vector into a plant is based onthe natural transformation system of Agrobacterium (see e.g., Horsch etal., 1985). A. tumefaciens and A. rhizogenes are plant pathogenic soilbacteria that genetically transform plant cells. The Ti and Ri plasmidsof A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant (see e.g., Kado,1991). Methods of introducing expression vectors into plant tissueinclude the direct infection or co-cultivation of plant cells withAgrobacterium tumefaciens (Horsch et al., 1985). Descriptions ofAgrobacterium vectors systems and methods for Agrobacterium-mediatedgene transfer provided by Gruber & Crosby, 1993, Moloney et al., 1989,and U.S. Pat. No. 5,591,616. General descriptions of plant expressionvectors and reporter genes and transformation protocols and descriptionsof Agrobacterium vector systems and methods for Agrobacterium-mediatedgene transfer can be found in Gruber & Crosby, 1993. General methods ofculturing plant tissues are provided for example by Miki et al., 1993and by Phillips et al., 1988. A reference handbook for molecular cloningtechniques and suitable expression vectors is Sambrook & Russell, 2001.

Another method for introducing an expression vector into a plant isbased on microprojectile-mediated transformation wherein DNA is carriedon the surface of microprojectiles. The expression vector is introducedinto plant tissues with a biolistic device that accelerates themicroprojectiles to speeds of 300 to 600 m/s which is sufficient topenetrate plant cell walls and membranes (see e.g., Sanford et al.,1987; Klein et al., 1988; Sanford, 1988; Sanford, 1990; Klein et al.,1992; Sanford et al., 1993). Another method for introducing DNA toplants is via the sonication of target cells (see Zhang et al., 1991).Alternatively, liposome or spheroplast fusion can be used to introduceexpression vectors into plants (see e.g., Deshayes et al, 1985 andChristou et al., 1987). Direct uptake of DNA into protoplasts usingCaCl₂ precipitation, polyvinyl alcohol, or poly-L-ornithine has alsobeen reported (see e.g., Hain et al. 1985 and Draper et al., 1982).Electroporation of protoplasts and whole cells and tissues has also beendescribed (D'Halluin et al., 1992 and Laursen et al., 1994).

Other well known techniques such as the use of BACs, wherein parts ofthe melon genome are introduced into bacterial artificial chromosomes(BACs), i.e., vectors used to clone DNA fragments (100- to 300-kb insertsize; average, 150 kb) in Escherichia coli cells, based on naturallyoccurring F-factor plasmid found in the bacterium E. coli. (Zhao &Stodolsky, 2004) can be employed for example in combination with theBIBAC system (Hamilton, 1997) to produce transgenic plants.

Following transformation of melon target tissues, expression of theabove described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, using standardregeneration and selection methods.

V. PRODUCTION OF FOM RACE 1,2-RESISTANT MELON PLANTS BY NON-TRANSGENICMETHODS

In some embodiments for producing an FOM race 1,2-resistant melon plant,protoplast fusion can be used for the transfer of nucleic acids from adonor plant to a recipient plant. Protoplast fusion is an induced orspontaneous union, such as a somatic hybridization, between two or moreprotoplasts (cells of which the cell walls are removed by enzymatictreatment) to produce a single bi- or multi-nucleate cell. The fusedcell, which can even be obtained with plant species that cannot beinterbred in nature, is tissue cultured into a hybrid plant exhibitingthe desirable combination of traits. More specifically, a firstprotoplast can be obtained from a melon plant or other plant line thatexhibits resistance to infection by FOM race 1,2. A second protoplastcan be obtained from a second melon or other plant variety, preferably amelon line that comprises commercially valuable characteristics, suchas, but not limited to disease resistance, insect resistance, valuablefruit characteristics, and the like. The protoplasts are then fusedusing traditional protoplast fusion procedures, which are known in theart.

Alternatively, embryo rescue can be employed in the transfer of anucleic acid comprising one or more QTLs as described herein from adonor plant to a recipient plant. Embryo rescue can be used as aprocedure to isolate embryo's from crosses wherein plants fail toproduce viable seed. In this process, the fertilized ovary or immatureseed of a plant is tissue cultured to create new plants (Pierik, 1999).

The presently disclosed subject matter also relates to methods forproducing an FOM race 1,2-resistant melon plant comprising performing amethod for detecting the presence of a quantitative resistance locus(QTL) associated with resistance to FOM race 1,2 in a donor melon plantaccording to the presently disclosed subject matter as described above,and transferring a nucleic acid sequence comprising at least one QTLthus detected, or a FOM race 1,2 resistance-conferring part thereof,from the donor plant to a FOM race 1,2-susceptible recipient melonplant. The transfer of the nucleic acid sequence can be performed by anyof the methods previously described herein.

An exemplary embodiment of such a method comprises the transfer byintrogression of the nucleic acid sequence from a FOM race 1,2-resistantdonor melon plant into a FOM race 1,2-susceptible recipient melon plantby crossing the plants. This transfer can thus suitably be accomplishedby using traditional breeding techniques. QTLs are introgressed in someembodiments into commercial melon varieties using marker-assistedselection (MAS) or marker-assisted breeding (MAB). MAS and MAB involvesthe use of one or more of the molecular markers for the identificationand selection of those offspring plants that contain one or more of thegenes that encode for the desired trait. In the context of the presentlydisclosed, subject matter, such identification and selection is based onselection of QTLs of the presently disclosed subject matter or markersassociated therewith. MAS can also be used to develop near-isogeniclines (NIL) harboring the QTL of interest, allowing a more detailedstudy of each QTL effect and is also an effective method for developmentof backcross inbred line (BIL) populations (see e.g., Nesbitt &Tanksley, 2001; van Berloo et al., 200.1). Melon plants developedaccording to these embodiments can advantageously derive a majority oftheir traits from the recipient plant, and derive FOM race 1,2resistance from the donor plant.

As discussed hereinabove, traditional breeding techniques can be used tointrogress a nucleic acid sequence encoding for FOM race 1,2 resistanceinto a FOM race 1,2-susceptible recipient melon plant. In someembodiments, a donor melon plant that exhibits resistance to FOM race1,2 and comprising a nucleic acid sequence encoding for FOM race 1,2resistance is crossed with a FOM race 1,2-susceptible recipient melonplant that in some embodiments exhibits commercially desirablecharacteristics, such as, but not limited to, disease resistance, insectresistance, valuable fruit characteristics, and the like The resultingplant population (representing the F1 hybrids) is then self-pollinatedand set seeds (F2 seeds). The F2 plants grown from the F2 seeds are thenscreened for resistance to FOM race 1,2. The population can be screenedin a number of different ways.

First, the population can be screened using a traditional diseasescreen. Such disease screens are known in the art. In some embodiments,a quantitative bioassay is used. Second, marker-assisted selection canbe performed using one or more of the herein-described molecular markersto identify those progeny that comprise a nucleic acid sequence encodingfor FOM race 1,2. Other methods, referred to hereinabove by methods fordetecting the presence of a QTL, can be used. Also, marker-assistedselection can be used to confirm the results obtained from thequantitative bioassays, and therefore, several methods can also be usedin combination.

Inbred FOM race 1,2-resistant melon plant lines can be developed usingthe techniques of recurrent selection and backcrossing, selfing, and/ordihaploids, or any other technique used to make parental lines. In amethod of recurrent selection and backcrossing, FOM race 1,2 resistancecan be introgressed into a target recipient plant (the recurrent parent)by crossing the recurrent parent with a first donor plant, which differsfrom the recurrent parent and is referred to herein as the“non-recurrent parent”. The recurrent parent is a plant that isnon-resistant or has a low level of resistance to FOM race 1,2 andpossesses commercially desirable characteristics, such as, but notlimited to (additional) disease resistance, insect resistance, valuablefruit characteristics, and the like. In some embodiments, thenon-recurrent parent exhibits FOM race 1,2 resistance and comprises anucleic acid sequence that encodes for FOM race 1,2 resistance. Thenon-recurrent parent can be any plant variety or inbred line that iscross-fertile with the recurrent parent.

The progeny resulting from a cross between the recurrent parent andnon-recurrent parent are backcrossed to the recurrent parent. Theresulting plant population is then screened for the desiredcharacteristics, which screening can occur in a number of differentways. For instance, the population can be screened using phenotypicpathology screens or quantitative bioassays as known in the art.Alternatively, instead of using bioassays, marker-assisted selection(MAS) can be performed using one or more of the hereinbefore describedmolecular markers, hybridization probes, or polynucleotides to identifythose progeny that comprise a nucleic acid sequence encoding for FOMrace 1,2 resistance. Also, MAS can be used to confirm the resultsobtained from the quantitative bioassays. In some embodiments, themarkers defined herein are suitable to select proper offspring plants bygenotypic screening.

Following screening, the F1 hybrid plants that exhibit a FOM race1,2-resistant phenotype or, in some embodiments, genotype and thuscomprise the requisite nucleic acid sequence encoding for FOM race 1,2resistance, are then selected and backcrossed to the recurrent parentfor a number of generations in order to allow for the melon plant tobecome increasingly inbred. This process can be performed for two,three, four, five, six, seven, eight, or more generations. In principle,the progeny resulting from the process of crossing the recurrent parentwith the FOM race 1,2-resistant non-recurrent parent are heterozygousfor one or more genes that encode FOM race 1,2 resistance.

In general, a method of introducing a desired trait into a hybrid melonvariety can comprise:

-   -   (a) crossing an inbred melon parent with another melon plant        that comprises one or more desired traits, to produce F1 progeny        plants, wherein the desired trait is FOM race 1,2 resistance;    -   (b) selecting the F1 progeny plants that have the desired trait        to produce selected F1 progeny plants, in some embodiments using        molecular markers as defined herein;    -   (c) backcrossing the selected progeny plants with the inbred        melon parent plant to produce backcross progeny plants;    -   (d) selecting for backcross progeny plants that have the desired        trait and morphological and physiological characteristics of the        inbred melon parent plant, wherein the selection comprises the        isolation of genomic DNA and testing the DNA for the presence of        at least one molecular marker for QTL1, QTL2, QTL3, QTL4, and/or        QTL5, in some embodiments as described herein;    -   (e) repeating steps (c) and (d) two or more times in succession        to produce selected third or higher backcross progeny plants;    -   (f) optionally selfing selected backcross progeny in order to        identify homozygous plants; and    -   (g) crossing at least one of the backcross progeny or selfed        plants with another inbred melon parent plant to generate a        hybrid melon variety with the desired trait and all of the        morphological and physiological characteristics of hybrid melon        variety when grown in the same environmental conditions.

As indicated, the last backcross generation can be selfed in order toprovide for homozygous pure breeding (inbred) progeny for FOM race 1,2resistance. Thus, the result of recurrent selection, backcrossing, andselfing is the production of lines that are genetically homogenous forthe genes associated with FOM race 1,2 resistance, and in someembodiments as well as for other genes associated with traits ofcommercial interest.

VI. FOM RACE 1,2-RESISTANT MELON PLANTS AND SEEDS

The goal of plant breeding is to combine in a single variety or hybridvarious desirable traits. For commercial crops, these traits can includeresistance to diseases and insects, tolerance to heat and drought,reducing the time to crop maturity, greater yield, and better agronomicquality. Uniformity of plant characteristics such as germination andstand establishment, growth rate, maturity, and plant height can also beof importance.

Commercial crops are bred through techniques that take advantage of theplant's method of pollination. A plant is self-pollinated if pollen fromone flower is transferred to the same or another flower of the sameplant. A plant is sib pollinated when individuals within the same familyor line are used for pollination. A plant is cross-pollinated if thepollen comes from a flower on a different plant from a different familyor line.

Plants that have been self-pollinated and selected for type for manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny. A cross between twodifferent homozygous lines produces a uniform population of hybridplants that may be heterozygous for many gene loci. A cross of twoplants each heterozygous at a number of gene loci will produce apopulation of heterogeneous plants that differ genetically and will notbe uniform.

The development of a hybrid melon variety in a melon plant breedingprogram can, in some embodiments, involve three steps: (1) the selectionof plants from various germplasm pools for initial breeding crosses; (2)the selling of the selected plants from the breeding crosses for severalgenerations to produce a series of inbred lines, which, individuallybreed true and are highly uniform; and (3) crossing a selected inbredline with an unrelated inbred line to produce the hybrid progeny (F1).After a sufficient amount of inbreeding successive filial generationswill merely serve to increase seed of the developed inbred. In someembodiments, an inbred line comprises homozygous alleles at about 95% ormore of its loci.

An important consequence of the homozygosity and homogeneity of theinbred lines is that the hybrid created by crossing a defined pair ofinbreds will always be the same. Once the inbreds that create a superiorhybrid have been identified, a continual supply of the hybrid seed canbe produced using these inbred parents and the hybrid melon plants canthen be generated from this hybrid seed supply.

A FOM race 1,2-resistant melon plant, or a part thereof, obtainable by amethod of the presently disclosed subject matter is an aspect of thepresently disclosed subject matter.

Another aspect of the presently disclosed subject matter relates to aFOM race 1,2-resistant melon plant, or part thereof, comprising the QTLsin any configuration as described in detail above wherein at least oneof the QTLs is not in its natural genetic background. The FOM race1,2-resistant melon plants of the presently disclosed subject matter canbe of any genetic type such as inbred, hybrid, haploid, dihaploid, ortransgenic. Further, the plants of the presently disclosed subjectmatter can be heterozygous or homozygous for the resistance traits (insome embodiments, homozygous). Although the QTLs of the presentlydisclosed subject matter, as well as resistance-conferring partsthereof, can be transferred to any plant in order to provide for a FOMrace 1,2-resistant plant, the methods and plants of the presentlydisclosed subject matter are in some embodiments related to plants ofthe species Cucumis melo.

The FOM race 1,2-resistant inbred melon lines described herein can beused in additional crossings to create FOM race 1,2-resistant hybridplants. For example, a first FOM race 1,2-resistant inbred melon plantof the presently disclosed subject matter can be crossed with a secondinbred melon plant possessing commercially desirable traits such as, butnot limited to, disease resistance, insect resistance, desirable fruitcharacteristics, and the like. In some embodiments, this second inbredmelon line is FOM race 1,2-resistant. In some embodiments, this line ishomozygous for one or more of QTL1-QTL5, in order for this recessivetrait to be expressed in the hybrid offspring plants.

Another aspect of the presently disclosed subject matter relates to amethod of producing seeds that can be grown into FOM race 1,2-resistantmelon plants. In some embodiments, the method comprises providing a FOMrace 1,2-resistant melon plant of the presently disclosed subjectmatter, crossing the FOM race 1,2-resistant plant with another melonplant, and collecting seeds resulting from the cross, which whenplanted, produce FOM race 1,2-resistant melon plants.

In some embodiments, the method comprises providing a FOM race1,2-resistant melon plant of the presently disclosed subject matter,crossing the FOM race 1,2-resistant plant with a melon plant, collectingseeds resulting from the cross, regenerating the seeds into plants,selecting FOM race 1,2-resistant plants by any of the methods describedherein, self-pollinating the selected plants for a sufficient number ofgenerations to obtain plants that are fixed for an allele that confersFOM race 1,2-resistance in the plants, backcrossing the plants thusproduced with melon plants having desirable phenotypic traits for asufficient number of generations to obtain melon plants that are FOMrace 1,2-resistant and have desirable phenotypic traits, and collectingthe seeds produced from the plants resulting from the last backcross,which when planted, produce melon plants which are FOM race1,2-resistant.

EXAMPLES

The following Examples provide illustrative embodiments. In light of thepresent disclosure and the general level of skill in the art, those ofskill will appreciate that the following Examples are intended to beexemplary only and that numerous changes, modifications, and alterationscan be employed without departing from the scope of the presentlyclaimed subject matter.

Example 1 Initial Plant Materials

Rupia F1 plants were obtained from Mikado Seed Growers Co. Ltd. andself-pollinated to produce an F2, several individuals of which wereself-pollinated to produce 18 F3 lines. Plants of these F3 lines(referred to herein as “MFR0029520”) were crossed with an F6 Charentaisline (referred to herein as “MFR0017377”), a line selected from theLunastar line (Nunhems Netherlands BV) to produce line referred toherein as “MFR0037489”, which was then backcrossed with the F6Charentais line MFR0017377 to obtain single backcross (BC1) linereferred to herein as “MFR0039432”. Double haploidization was performedon a plant of BC1 line MFR0039432, and 29 double haploid lines wereobtained. One such double haploid, referred to herein as “03MFR001795”,was selected for good resistance to F. oxysporum f.sp. melonis race 1,2in climatic chambers according to the phenotypic evaluation described inthe EXAMPLE 3 below. This male plant had green flesh color. The03MFR001795 double haploid line was crossed with a second Charentais F6line MFR0040308 to produce a hybrid referred to herein as “02MFR005456”.This hybrid was selfed to produce F2 seed.

Example 2 Recombinant Inbred Lines Development Using the Single SeedDescent (SSD) Method

200 F2 plants from the selfing of 02MFR005456 were self-pollinated and199 F3 lines were obtained. All 199 F3 lines were sown, and one plantper F3 was transplanted and self-pollinated to generate 196 F4 lines.These 196 F4 lines were sown, and one plant per F4 was transplanted andself-pollinated to generate 194 F5 lines. These 194 F5 lines were sown,and one plant per F5 was transplanted and self-pollinated to generate183 F6 lines. These 183 F6 lines were sown, and one plant per F6 wastransplanted and self-pollinated to generate 177 F7 lines.

Leaf tissue from each F6 plant was collected and used for DNA extractionsand genotyping.

F7 seeds that were produced by each F6 plant were harvested, and all F7seeds harvested were kept separated by F6 plant of origin, therebyconstituting 177 F7 families. The 177 F7 families were evaluated for F.oxysporum f.sp. melonis race 1,2 resistance in the sand test.

Example 3 Phenotypic Evaluation

Fungal Strain.

A yellowing isolate of Fusarium oxysporum f.sp. melonis race 1,2 wasused for the phenotypic evaluations of the RIL population and also oflines and hybrids from breeding programs. The strain was maintained onPetri dishes with agar medium S under controlled temperature at 20° C.Medium S contains 1 g/l Ca(NO₃)₂, 0.25 g/l KNO₃, 0.25 g/l MgSO₄, 0.125g/l KH₂PO₄, 0.125 g/l K₂HPO₄, 0.05 g/l citric acid, 5 g/1 malt, and 50g/l sucrose. For solid medium, 25 g of agar was added per liter ofmedium S, and the material was autoclaved to sterility, cooled, andpoured onto Petri dishes.

A monthly subculture was made from a small piece of agar containingmycelium into a new sterile Petri dish. Active cultures were obtained byplacing a small piece of agar containing mycelium in a flask containing400 ml of medium S and incubating on a rotary shaker at 130 rpm for 3days at 21° C.

RIL Disease Evaluation.

Among the 177 RILs, 154 lines and controls were evaluated for resistanceto F. oxysporum f.sp. melons race 1,2 after artificial inoculation.Three independents experiments were carried out. In each experiment, 10plants of each RIL were evaluated in a complete randomized design with 3blocks.

Seeds were sown in trays with adapted compost for sowing. Trays wereincubated in climatic chambers with a photoperiod of 15 h/9 h(day/night). The temperature during the day was 24° C.±2° C. with aluminosity of 10,000 lux, and during the night the temperature was 18°C.±2° C.

The inoculation was carried out after 7 days of growth. Seedlings wereremoved from the compost and roots were washed with water before beingincubated for 10 minutes in a solution containing 1×10⁶ spores/ml of ayellowing strain of F. oxysporum f.sp. melonis race 1,2. Thereafter,seedlings were transplanted in sandy trays with 5 plants per row. Eachtray included appropriate controls. Trays were incubated in a climaticchamber with a 12 h/12 h light/dark cycle at a temperature of 22° C./18°C.±2° C. During the light cycle the luminosity was 5000 lux. Trays werewatered each day during the first weeks, and then a nutritive solutionwas added every other watering until the end of the evaluation.(Liquoplant Bleu from Plantin, Courthezon, France; with the followingNPK composition: 2.5 (whole nitrogen)−5 (P₂O₅)−2.5 (K₂O)−0.75(MgO)+oligo-elements. The solution is diluted to obtain anelectro-conductivity of 2 and a pH of 6.5).

The first symptoms (i.e., yellowing on cotyledons) appeared after day 7post-inoculation. After days 15, 20, and 25 post-inoculation, theevaluation of symptoms was assessed on infected leaves using asemi-quantitative rating scale from 1 to 5 as follows:

1=no symptoms;

2=yellowing of the cotyledons or the first leaf;

3=yellowing of two leaves;

4=yellowing of three or more leaves;

5=death of plant.

The susceptible plants showed a yellowing on cotyledons after 7 days,stop growing, and died within a few days. The intermediate resistantplants showed slowed growth and symptoms appeared on new leaves. Theresistant plants did not show any symptoms on leaves and grew at anormal rate.

25 days post-inoculation, all plants were scored on thesemi-quantitative rating scale (1-5) above. The disease scores werecalculated for each RIL using a mean by line, by block, and byexperiment. For each RIL, a mean disease score of each plant wasrecorded.

Lines and Hybrids Evaluation in a Breeding Program.

Lines were evaluated under similar conditions as indicated for the RILpopulation; but the trial design was simplified by testing 20 plants perline. the evaluation of symptoms was assessed on infected leaves using,the same semi-quantitative 1-9 rating scale defined below for hybridsevaluation

Hybrid evaluation was performed on 3 replicates of 8 plants per hybrid.Seeds were sown in trays with adapted compost for sowing. Trays wereincubated in climatic chambers with a photoperiod of 15 h/9 h(day/night). The temperature during the day was 24° C.±2° C. with aluminosity of 10,000 lux, and during the night the temperature was 18°C.±2° C.

Six (6) days after sowing, seedlings were transplanted into trayscontaining 96 conical pots each with a diameter of 6 cm. Trays wereincubated in a climatic chamber (12 h/12 h day/night) at 22° C./18°C.±2° C. During the day cycle, the luminosity was 5000 lux. Trays werewatered each day during the first weeks then a nutritive solution wasadded as set forth hereinabove.

One week later (i.e., at day 15 post-sowing), when the first true leafmeasured a few cm in length, plants were inoculated by soaking the trayinto a spore solution containing 1×10⁵ spores/ml for 10 minutes.

The first symptoms (i.e., a yellowing on cotyledons) appeared after day10 post-inoculation. After days 15, 20, and 25 post-inoculation, theevaluation of symptoms was assessed on infected leaves using asemi-quantitative rating scale from 1 to 3 as follows:

1=Resistant (no symptoms)

2=Intermediate Resistance (yellowing of the cotyledons or the firstleaf)

3=Susceptible (yellowing of two or more leaves)

The disease score at the third scoring date (day 25 post-inoculation)was assessed for each plant of the hybrid. To stretch the scale and tocompare hybrids, a score of from 1 to 9 was calculated for each hybridusing the following calculation:Store=((X×9)+(Y×5)+(Z×1))/(X+Y+Z),

wherein:

X=number of plants for an hybrid with a score equal to 3;

Y=number of plants for an hybrid with a score equal to 2; and

Z=number of plants for an hybrid with a score equal to 1.

Example 4 Genotyping and QTL Mapping

DNA was extracted from F6 leaves and DNA samples were genotyped using 82polymorphic SSRs. Several hundred SSRs covering the entire melon genomehad been previously run on the two parents of this segregatingpopulation, MC7752, in order to identify the polymorphic ones. Amolecular marker map was constructed using MapMaker (Lander et al.,1987) and JoinMap (Stam, 1993) software. Joint-analysis of genotypic andphenotypic data was performed using the software QTL Cartographer andPLABQTL (Utz & Melchinger, 1996). Five QTLs were identified for Fusariumoxysporum f.sp. melonis race 1,2 resistance on chromosomes 3, 6, 7, 9,and 10. These QTLs are characterized by their position on the geneticmap and their additive and dominant effects. Positions were defined asgenetic distances between the most likely position of the QTLs (usuallythe position of the peak LOD score value) and linked marker loci (incentiMorgans). Additive effects were defined as deviations from the meanand were expressed in the same units as the trait they refer to (i.e., 1to 5 scale, where 1 is fully resistant and 5 is fully susceptiblefamily). Additive values defined which of the two parental lines carriedthe favorable allele at the QTL. In this case, positive additive valuesidentified for all five QTLs meant that the 03MFR001795 parent carriedthe favorable alleles for these QTLs.

The position of the five QTLs identified relative to neighboringmarkers, along with their effects and favorable alleles, are presentedin Table 1. These QTLs were thus the selection targets for thedevelopment of new Fusarium oxysporum f.sp. melonis race 1,2 resistantlines.

TABLE 1 QTLs for F. oxysporum f.sp melonis Race 1, 2 Resistance andLinked Markers QTL QTL QTL Effect QTL Begin Position End (Add Linked #Chrom (cM)* (cM)* (cM)* Value) Markers** 1 3 81.1 90.2 94.4 0.34 3.1,3.2 2 6 6.6 13.6 28.8 0.32 6.1, 6.2 3 7 1.3 9.3 19.1 0.23 7.1, 7.2 4 93.2 21.1 23.7 0.54 9.1, 9.2, 9.3 5 10 n.d ~43 n.d 0.22 10.1, 10.2 *thesevalues are approximate and are based on the mapping population employedherein. One of ordinary skill in the art would recognize that indifferent mapping populations, the absolute positions might vary,although the placement of the markers on chromosomes relative to eachother would not be expected to differ. n.d.: not determined. **For QTLs1, 2, 3, and 5, markers 3.1, 6.1, 7.1, and 10.1 map to the positionlisted under the heading “QTL Begin” for the corresponding QTL, andmarkers 3.2, 6.2, 7.2, and 10.2 map to the position listed under theheading “QTL End”. With respect to QTL4, markers 9.1, 9.2, and 9.3 mapto the positions listed under the headings “QTL Begin”, “QTL Position”,and “QTL END”, respectively.

In Table 1, each QTL has been assigned an arbitrary number 1-5. Thefollowing information is presented for each QTL: the chromosome on whichit is located, its most likely position on that chromosome (i.e., theposition with the highest LOD score), the beginning and end of itsconfidence interval, its effect (additive value) as characterized by thedifference between the effect of the allele from 03MFR001795 and that ofthe allele from MFR0040308, and markers linked to the QTL (and thereforediagnostic of the allele present at the QTL).

For example, the first QTL for F. oxysporum f.sp. melonis race 1,2resistance was located on chromosome 3, with a most likely position at90.2 cM and with a confidence interval ranging from 80.7 cM to 99.4 cM.The effect of the QTL is 0.34, which means that the allele 03MFR001795increased resistance by 0.34 compared to the allele from MFR0040308.

Example 5 Determination of Allele Characteristics (Amplified FragmentSize) by PCR

In all identified QTLs, the favorable allele came from 03MFR001795(deposited with NCIMB Ltd. under the terms of the Budapest Treaty on 27Apr. 2007 as Accession No. 41478). A PCR assay and/or the sequencingassay set forth in EXAMPLE 6 were employed to screen DNA isolated fromplants for the presence of these alleles. This information was used toselect individuals during the marker-based selection process, theobjective of which is to maximize the number of favorable alleles for F.oxysporum f.sp. melonis race 1,2 resistance present in one individual.

Three (3) μl of extracted plant DNA to be tested (concentration equals 2ng/μl) was distributed in to wells of 384-well plates. 3 μl of PCR mix Awas also added to the wells. The composition of PCR mix A was 1×PCRbuffer containing 1.65 mM MgCl₂, each dNTP at 62.5 μM, 0.033 units/μlInvitrogen PLATINUM® Taq polymerase (Invitrogen Corp., Carlsbad, Calif.,United States of America), and M13/forward and reverse primers at 412 nMeach. The sequences of the primers employed are as follows:

QTL 1 (Chromosome 3):

-   -   3.1: M13/forward primer GTTTTCCCAGTCACGACGCGTCATAGCG TACTTAGC        (SEQ ID NO: 23/SEQ ID NO: 1) and reverse primer        ATTTGTTTTGCCATTTCTG (SEQ ID NO: 2). Size of fragment from        desired allele: 339 basepairs (bp).    -   3.2: M13/forward primer GTTTTCCCAGTCACGACCCAAATCGA AACAAAAGTC        (SEQ ID NO: 23/SEQ ID NO: 3) and reverse primer        TGTTAGATTTGTTGCAGGC (SEQ ID NO: 4). Size of fragment from        desired allele: 320 bp.        QTL 2 (Chromosome 6):    -   6.1: M13/forward primer GTTTTCCCAGTCACGACACAAAATGGT AATGAAAACTTG        (SEQ ID NO: 23/SEQ ID NO: 5) and reverse primer        AACAAGAAAGCTACCACGC (SEQ ID NO: 6). Size of fragment from        desired allele: 254 bp.    -   6.2: M13/forward primer GTTTTCCCAGTCACGACCCCATGAAAG AAAATGGAG        (SEQ ID NO: 23/SEQ ID NO: 7) and reverse primer        TTCATCTTCCATCAAACCC (SEQ ID NO: 8). Size of fragment from        desired allele: 206 bp.        QTL 3 (Chromosome 7):    -   7.1: M13/forward primer GTTTTCCCAGTCACGACTAGCTTGAACTT CGTCCTG        (SEQ ID NO: 23/SEQ ID NO: 9) and reverse primer        GAAGCGTACTCCCTATTGC (SEQ ID NO: 10). Size of fragment from        desired allele: 238 bp.    -   7.2: M13/forward primer GTTTTCCCAGTCACGACGGCAGTAAAT GACCATGAC        (SEQ ID NO: 23/SEQ ID NO: 11) and reverse primer        GGTGACGAACAAACTGAAG (SEQ ID NO: 12). Size of fragment from        desired allele: 246 bp.        QTL 4 (Chromosome 9):    -   9.1: M13/forward primer GTTTTCCCAGTCACGACTAGCAAAC GACAACTAGGC        (SEQ ID NO: 23/SEQ ID NO: 13) and reverse primer        GTGGAAAAGAGAGGAAAGG (SEQ ID NO: 14). Size of fragment from        desired allele: 346 bp.    -   9.2: M13/forward primer GTTTTCCCAGTCACGACCCCCTCTTAT CTTTTCCTG        (SEQ ID NO: 23/SEQ ID NO: 15) and reverse primer        CATCAAGAAGTCACGGAAG (SEQ ID NO: 16). Size of fragment from        desired allele: 296 bp.    -   9.3: M13/forward primer GTTTTCCCAGTCACGACCCAAAGTAAAAG TGAAGTCC        (SEQ ID NO: 23/SEQ ID NO: 17) and reverse primer        CTTGAAATGAATTTGAGGTG (SEQ ID NO: 18). Size of fragment from        desired allele: 164 bp.        QTL 5 (Chromosome 10):    -   10.1: M13/forward primer GTTTTCCCAGTCACGACTTCTGATCAAC GACGAAG        (SEQ ID NO: 23/SEQ ID NO: 19) and reverse primer        GAAACAAAAGCCTCCATTG (SEQ ID NO: 20). Size of fragment from        desired allele: 263 bp.    -   10.2: M13/forward primer GTTTTCCCAGTCACGACACCCACCATG CATTCTAAC        (SEQ ID NO: 23/SEQ ID NO: 21) and reverse primer        GAGCCAGTTGGGGTTTTAG (SEQ ID NO: 22). Size of fragment from        desired allele: 268 bp.

The PCR amplification was conducted with in a GENEAMP® PCR System 9700thermocycler (Applied Biosystems, Inc. Foster City, Calif., UnitedStates of America) employing the following steps: an initialdenaturation of 2 minutes at 94° C.; 40 cycles of 0 seconds at 94° C.followed by 0 seconds at 54° C. and 5 seconds at 72° C. (i.e., 40 cyclesof ramping up to 94° C. and down to 54° C., each with no hold time,before a 5 second extension step at 72° C.). Amplified products wereseparated on agarose gels using high resolution agarose(ULTRAPURE®Agarose 1000 from Invitrogen Corp.) at a concentration of 3%in 1× Tris-borate EDTA (TBE). Electrophoresis was conducted at 400 voltsfor approximately 1 hour. PCR amplification products were observed afteragarose separation using ethidium bromide and viewing under UV light.

Example 6 Determination of Allele Characteristics (Amplified FragmentSize) Using a Sequencer

Five (5) μl of DNA at a concentration of 2 ng/μl was distributed intowells of 384-well plates. 5 μl of PCR mix B was also added to the wells.The composition of PCR mix B was 1×PCR buffer containing 1.65 mM MgCl₂,each dNTP at 200 μM, 0.033 units/μl Invitrogen PLATINUM® Taq polymerase(Invitrogen Corp.), a M13/forward primer at 800 nM, a reverse primer at600 nM, and a fluorescent M13 probe at 600 nM. Primer sequences were asshown in EXAMPLE 5. The fluorescent M13 probe included a fluorescentlabel attached to its 5′ end and a nucleotide sequence that specificallyhybridized to SEQ ID NO: 23, allowing the fluorescent probe to beemployed for detecting the size of the amplified fragments in thesequencer.

PCR amplification was conducted using a GENEAMP® PCR System 9700thermocycler (Applied Biosystems) and the following steps: an initialdenaturation step of 2 minutes at 94° C.; 40 cycles of 15 seconds at 94°C. followed by 45 seconds at 54° C.; and a final extension of 2 minutesat 72° C. PCR amplification products were denatured with formamide for 3minutes at 96° C. before being separated on an ABI PRISM® 3700 sequencer(Applied Biosystems). Migration in the sequencer took place incapillaries filled with polymer POP-6™ (Applied Biosystems) in 1×TBE.The sizes of the PCR amplification fragments were determined softwareGENESCAN® and GENOTYPER® software (Applied Biosystems).

Example 7 Introgression of Resistance in Charentais Breeding MaterialUsing Pathology Tests: 06MFR006171 Male Parental Line Example

Alleles associated with resistance to F. oxysporum f.sp. melonis race1,2 present in Rupia have been introgressed into Charentais breedingmaterial by rescuing resistant plants after sand tests and backcrossingthem to Charentais breeding lines. The Charentais melon has orange fleshand is climacteric at maturity.

A Rupia F1 hybrid was crossed in September 2000 to Charentais dihaploidline MFR0038049 to generate Cross RUPIA×MFR0038049. CrossRUPIA×MFR0038049 was placed in a sand test. 36 surviving plants wererescued, grown, and self pollinated in the greenhouse in April 2001.Each plant was backcrossed to dihaploid Charentais line MFR0038049 toobtain 33 BC1 individuals.

These BC1 individuals were tested in a sand test and 2 surviving plantswere rescued, grown, and self-pollinated in the greenhouse to obtain 2F2BC1 lines.

One such F2BC1 line (referred to herein as “MFR0043937”) was tested in asand test and 3 surviving plants were rescued, grown, andself-pollinated in the greenhouse to obtain an F3 line (referred toherein as “02MFR002965”) that was heterozygous (orange/green) for fleshcolor.

F3 line 02MFR002965 (heterozygous for flesh color) was tested in a sandtest and 23 surviving plants were rescued, grown, and self-pollinated inthe greenhouse. These 23 plants were crossed to an F4 Charentais line(referred to herein as “MFR0043194”) to produce a series of progenyplants.

Individuals of these progeny plants were tested in a sand test and 5surviving plants were rescued, grown, and self-pollinated in thegreenhouse. Each plant was crossed with Charentais line 02MFR010858 togenerate further progeny.

Individuals of these further progeny were tested in a sand test and 22surviving plants were rescued, grown, and self-pollinated in thegreenhouse. 22 F2 lines were obtained and tested for Fusarium oxysporumf.sp. melon's race 1,2 resistance.

These 22 F2 lines were tested in a sand test and surviving plants wererescued, grown, and self-pollinated in the greenhouse. These F2 linessegregated for flesh color. 8 F3 were obtained and tested for Fusariumoxysporum f.sp. melonis race 1,2 resistance.

An individual F3 line was tested in a sand test and surviving plantswere rescued, grown, and self-pollinated in the greenhouse. This F3 linewas fixed for orange flesh color. 3 F4 lines were selected and tested ina sand tested for Fusarium oxysporum f.sp. melonis race 1,2 resistance.

An individual F4 line was tested in a sand test and surviving plantswere rescued, grown, and self-pollinated in the greenhouse. 15 F5 lineswere obtained. They were tested in a sand test for Fusarium oxysporumf.sp. melonis race 1,2 resistance.

An individual F5 line was used as a male to cross with susceptibleCharentais dihaploid line MFR0044433. The hybrid obtained was referredto as 05MFR013549.

The F5 line was also self-pollinated to generate an F6 line.

05MFR013549 was tested in a phytotron multicell test and showed goodresistance to Fusarium oxysporum f.sp. melonis race 1,2. 05MFR013549 wasalso evaluated in an open field protected by a small tunnel. The trialfield was strongly infected by Fusarium oxysporum f.sp. melonis race1,2. The hybrid 05MFR013549 showed an intermediate resistance toFusarium oxysporum f.sp. melonis race 1,2.

The F6 line was self pollinated and an F7 line was obtained. The F7 line(06MFR003787) was crossed with line MFR0044433. Hybrid 06MFR006171 wasobtained and was tested in a multicell test in a phytotron.

These breeding experiments, along with the results of testing for thepresence or absence of representative markers in QTLs 1 and 4, aresummarized in Tables 2 and 3.

TABLE 2 Types, Sources, and Flesh Colors of Representative Lines andHybrids MATID Type Source FLC  1. MFR0040308 Line Syngenta O  2.MFR0038049 Line Syngenta O  3. MFR0025266 Line Syngenta O  4. MFR0044433Line Syngenta O  5. 03MFR003030 Line Syngenta O  6. RUPIA Hybrid MikadoO  7. 03MFR001795 Line Syngenta G  8. 06MFR003786** Line Syngenta O  9.06MFR006172 Hybrid Syngenta O 10. 06MFR009993 Hybrid Syngenta O 11.06MFR003787 Line Syngenta O 12. 06MFR006171 Hybrid Syngenta O 13.06MFR009975 Hybrid Syngenta O 14. 06MFR004012 Line Syngenta O 15.06MFR006175 Hybrid Syngenta O 16. 06MFR005919 Line Syngenta O 17.06MFR009976 Hybrid Syngenta O 18. 06MFR009994 Hybrid Syngenta O 19.FIDJI Hybrid Gautier O 20. MANTA Hybrid Clause-Tezier O 21. AMADORAHybrid Syngenta O 22. MEHARI Hybrid Syngenta O

TABLE 3 Presence or Absence of Markers in QTL1 and QTL4 QTL1 QTL4 LineHybrid Markers Markers MATID test* test* 3.1 3.2 9.1 9.2 9.3  1.MFR0040308 6.6 — 0 0 0 0 0  2. MFR0038049 7.1 — 0 0 0 0 0  3. MFR00252668.5 — 0 0 0 0 0  4. MFR0044433 8.7 — 0 0 0 0 0  5. 03MFR003030 6.8 — 0 00 0 0  6. RUPIA 7.3 4.0 1 1 H H H  7. 03MFR001795 2.9 — 1 1 1 1 1  8.06MFR003786** 4.1 — 0 1 0 1 1  9. 06MFR006172 — 4.0 0 H 0 H H 10.06MFR009993 — 3.2 0 H 0 H H 11. 06MFR003787 4.9 — 0 1 0 1 1 12.06MFR006171 — 4.2 0 H 0 H H 13. 06MFR009975 — 2.2 0 H 0 H H 14.06MFR004012 2.9 — 0 1 0 1 1 15. 06MFR006175 — 5.2 0 H 0 H H 16.06MFR005919 6.7 — 0 0 0 0 0 17. 06MFR009976 — 7.7 0 0 0 0 0 18.06MFR009994 — 8.2 0 0 0 0 0 19. FIDJI 8.3 5.8 0 0 0 0 0 20. MANTA 5.72.6 0 0 0 0 0 21. AMADORA — 8.5 0 0 0 0 0 22. MEHARI — 8.8 0 0 0 0 0

Legend for Tables 2 and 3:

-   -   All hybrids and lines were in a Charentais background with the        exception of Rupia, which is a Japanese cultivar.    -   Rows 1-5: FOM race 1,2 susceptible recurrent lines; Row 6: Rupia        F1 FOM race 1,2 intermediate resistant from Mikado Seeds; Row 7:        FOM race 1,2 resistant donor of RIL mapping population; Rows        8-10: FOM race 1,2 donor line and corresponding hybrids; Rows        11-13: FOM race 1,2 donor line and corresponding hybrid; Rows        14-15: FOM race 1,2 donor line and corresponding hybrid; Rows        16-18: FOM race 1,2 donor line and corresponding hybrids; Rows        19-20: Other commercial hybrids with FOM race 1,2 intermediate        resistance levels; Rows 21-22: FOM race 1,2 susceptible hybrid        checks.    -   FLC: flesh color; O: orange; G: green;    -   *: Resistance to FOM race 1,2 scored on a scale of 1-9 as        described herein;    -   **: this line, 06MFR003786, is homozygous for QTL4 and yet has        orange flesh, indicative of a breaking of the linkage between        the green flesh color marker(s) present on chromosome 9 and        QTL4.    -   -: not applicable; 0: favored allele absent; 1: favored allele        present in homozygous state; H: favored allele present in        heterozygous state.

REFERENCES

All references listed below, as well as all references cited in theinstant disclosure, including but not limited to all patents, patentapplications and publications thereof, scientific journal articles, anddatabase entries (e.g., GENBANK® database entries and all annotationsavailable therein) are incorporated herein by reference in theirentireties to the extent that they supplement, explain, provide abackground for, or teach methodology, techniques, and/or compositionsemployed herein.

-   Altschul et al. (1990) J Mol Biol 215:403-10.-   Altschul et al. (1997) Nucleic Acids Res 25:3389-3402.-   Ausubel et al. (eds.) (1999) Short. Protocols in Molecular Biology    Wiley, New York, N.Y., United States of America.-   Christou et al. (1987) Proc Natl Acad Sci USA 84:3962-3966.-   Deshayes et al. (1985) EMBO J 4:2731-2737.-   Draper et al. (1982) Plant Cell Physiol 23, 451-458.-   Glick & Thompson (1993) Methods in Plant Molecular Biology and    Biotechnology, CRC Press, Boca Raton. Fla., United States of    America.-   Gruber & Crosby (1993) in Glick & Thompson (eds.) Methods in Plant    Molecular Biology and Biotechnology, CRC Press, Baton Rouge, La.,    United States of America, pages 89-119.-   Hain at al. (1985) Mol Gen Genet. 199:161-168.-   Hamilton (1997) Gene 200:107-116.-   Horsch et al. (1985) Science 227:1229-1231.-   Kado (1991) Crit Rev Plant Sci 10:1-32.-   Klein et al. (1988) Biotechnology 6:559-563.-   Klein at al. (1992) Bio/Technology 10:286-291.-   Lander et al. (1987) Genomics 1:174-181.-   Laursen et al, (1994) Plant Mol Biol 24:51-61.-   Miki et al. (1993). in Glick & Thompson (eds.) Methods in Plant    Molecular Biology & Biotechnology, CRC Press, Baton Rouge, La.,    United States of America, pages 67-88.-   Moloney et al. (1989) Plant Cell Rep 8:238-242.-   Nesbitt & Tanksley (2001) Plant Physiol 127:575-583.-   Paterson (1996) in Paterson (ed.) Genome Mapping in Plants. R. G.    Landes Company, Georgetown, Tex., United States of America, pages    41-54.-   Perchepied et al. (2005) Theor Appl Genet 111:65-74.-   Perin et al., (2002) Theor Appl Genet 104:1017-1034.-   Phillips et al. (1988) in Sprague & Dudley (eds.), Corn and Corn    Improvement, 3rd ed., American Society of Agronomy, Madison, Wis.,    United States of America, pages 345-387.-   Pierik (1999) In vitro Culture of Higher Plants, 4th edition.    Martinus Nijhoff. Publishers, Dordrecht, The Netherlands.-   Risser et al. (1976) Phytopathology 66:1105-1106.-   Sambrook & Russell (2001). Molecular Cloning: A Laboratory Manual,    Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y., United States of America.-   Sanford (1988). Trends Biotechnol 6:299-302.-   Sanford (1990) Physiologica Plantarum, 79:206-209.-   Sanford et al. (1987). J Particulate Sci Technol 5:27-37.-   Sanford et al., (1993) Meth Enzymol 217:483-509.-   Stam (1993) Plant J 3:739-744.-   Tijssen (1993) in Laboratory Techniques in Biochemistry and    Molecular Biology, Elsevier, New York, N.Y., United States of    America.-   U.S. Pat. Nos. 4,458,066; 5,591,616.-   Utz & Melchinger (1996) J Agricultural Genomics 2:1-5.-   Van Berloo et al. (2001) Mol Breeding 8:187-195.-   Zhang et al. (1991) Biotechnology. 9:996-997.-   Zhao & Stodolsky (2004) Bacterial Artificial Chromosomes. Methods in    Molecular Biology Vol. 255. Humana Press Inc. Totowa, N.J., United    States of America.-   Zietkiewicz et al. (1994) Genomics 20:176-183.

It will be understood that various details of the presently disclosedsubject matter may be changed without departing from the scope of thepresently disclosed subject matter. Furthermore, the foregoingdescription is for the purpose of illustration only, and not for thepurpose of limitation.

What is claimed is:
 1. A method for the production of an inbred melon plant adapted for conferring, in hybrid combination with a suitable second inbred, resistance to F. oxysporum f.sp. melonis (FOM) race 1,2, the method comprising: (a) selecting a first donor parental line possessing a desired FOM race 1,2 resistance and having a resistant locus mapping to linkage group 9 and mapped by one or more of the markers 9.1, 9.2, and 9.3; (b) crossing the first donor parent line with a second parental line in hybrid combination to produce a segregating plant population; (c) screening the segregating plant population for identified chromosomal loci of one or more genes associated with the resistance to FOM race 1,2; and (d) selecting plants from the population having the identified chromosomal loci for further screening until a line is obtained which is homozygous for resistance to FOM race 1,2 at sufficient loci to give resistance to FOM race 1,2 in hybrid combination.
 2. A method for producing melon plants which are resistant to F. oxysporum f.sp. melonis (FOM) race 1,2 occurring in melon, the method comprising: (e) providing a Cucumis melo plant which contains one or more alleles that confer resistance to FOM race 1,2, which alleles are present within one or more of five Quantitative Trait Loci QTL1-QTL5 on different chromosomes, wherein: (i) QTL1 is defined by the following markers: (1) a marker of about 322 basepairs (bp), wherein the marker corresponds to an amplification product generated by amplifying a Cucumis melo nucleic acid with a forward primer comprising a nucleotide sequence as set forth in SEQ ID NO: 1 and a reverse primer comprising a nucleotide sequence as set forth in SEQ ID NO: 2; and (2) a marker of about 303 bp, wherein the marker corresponds to an amplification product generated by amplifying a Cucumis melo nucleic acid with a forward primer comprising a nucleotide sequence as set forth in SEQ ID NO: 3 and a reverse primer comprising a nucleotide sequence as set forth in SEQ ID NO: 4; or any part of a DNA sequence as in 03MFR001795 linked within 1, 2, 5, or 10 cM to at least one of the markers of (1) and (2) conferring resistance to FOM race 1,2; (ii) QTL2 is defined by the following markers: (1) a marker of about 237 bp, wherein the marker corresponds to an amplification product generated by amplifying a Cucumis melo nucleic acid with a forward primer comprising a nucleotide sequence as set forth in SEQ ID NO: 5 and a reverse primer comprising a nucleotide sequence as set forth in SEQ ID NO: 6; and (2) a marker of about 189 bp, wherein the marker corresponds to an amplification product generated by amplifying a Cucumis melo nucleic acid with a forward primer comprising a nucleotide sequence as set forth in SEQ ID NO: 7 and a reverse primer comprising a nucleotide sequence as set forth in SEQ ID NO: 8, or any part of a DNA sequence as in 03MFR001795 linked within 1, 2, 5, or 10 cM to at least one of the markers of (1) and (2) conferring resistance to FOM race 1,2; (iii) QTL3 is defined by the following markers: (1) a marker of about 221 bp, wherein the marker corresponds to an amplification product generated by amplifying a Cucumis melo nucleic acid with a forward primer comprising a nucleotide sequence as set forth in SEQ ID NO: 9 and a reverse primer comprising a nucleotide sequence as set forth in SEQ ID NO: 10; and (2) a marker of about 229 bp, wherein the marker corresponds to an amplification product generated by amplifying a Cucumis melo nucleic acid with a forward primer comprising a nucleotide sequence as set forth in SEQ ID NO: 11 and a reverse primer comprising a nucleotide sequence as set forth in SEQ ID NO: 12, or any part of a DNA sequence as in 03MFR001795 linked within 1, 2, 5, or 10 cM to at least one of the markers of (1), and (2) conferring resistance to FOM race 1,2; (iv) QTL4 is defined by the following markers: (1) a marker of about 329 bp, wherein the marker corresponds to an amplification product generated by amplifying a Cucumis melo nucleic acid with a forward primer comprising a nucleotide sequence as set forth in SEQ ID NO: 13 and a reverse primer comprising a nucleotide sequence as set forth in SEQ ID NO: 14; (2) a marker of about 279 bp, wherein the marker corresponds to an amplification product generated by amplifying a Cucumis melo nucleic acid with a forward primer comprising a nucleotide sequence as set forth in SEQ ID NO: 15 and a reverse primer comprising a nucleotide sequence as set forth in SEQ ID NO: 16; and (3) a marker of about 147 bp, wherein the marker corresponds to an amplification product generated by amplifying a Cucumis melo nucleic acid with a forward primer comprising a nucleotide sequence as set forth in SEQ ID NO: 17 and a reverse primer comprising a nucleotide sequence as set forth in SEQ ID NO: 18, or any part of a DNA sequence as in 03MFR001795 linked within 1, 2, 5, or 10 cM to at least one of the markers of (1), (2), and (3) conferring resistance to FOM race 1,2; and (v) QTL5 is defined by the following markers: (1) a marker of about 246 bp, wherein the marker corresponds to an amplification product generated by amplifying a Cucumis melo nucleic acid with a forward primer comprising a nucleotide sequence as set forth in SEQ ID NO: 19 and a reverse primer comprising a nucleotide sequence as set forth in SEQ ID NO: 20; and (2) a marker of about 251 bp, wherein the marker corresponds to an amplification product generated by amplifying a Cucumis melo nucleic acid with a forward primer comprising a nucleotide sequence as set forth in SEQ ID NO: 21 and a reverse primer comprising a nucleotide sequence as set forth in SEQ ID NO: 22, or any part of a DNA sequence as in 03MFR001795 linked within 1, 2, 5, or 10 cM to at least one of the markers of (1) and (2) conferring resistance to FOM race 1,2; and (f) crossing the Cucumis melo plant provided in step (a) with Cucumis melo culture breeding material to produce one or more progeny individuals, whereby one or more melon plants which are resistant to F. oxysporum f.sp. melonis (FOM) race 1,2 occurring in melon are produced.
 3. The method of claim 2, further comprising: (a) collecting the seeds resulting from the cross in step (b); (b) regenerating the seeds into plants; (c) evaluating the plants of step (d) for resistance to FOM race 1,2; and (d) identifying and selecting plants which are resistant to the FOM race 1,2.
 4. The method of claim 2, wherein the Cucumis melo plant provided in step (a) is 03MFR001795 or an ancestor or descendent thereof.
 5. A method for identifying a first Cucumis melo plant or germplasm that displays resistance, improved resistance, or reduced susceptibility to FOM race 1,2, the method comprising detecting in the first Cucumis melo plant or germplasm at least one allele of one or more marker locus that is associated with the tolerance, improved tolerance, reduced susceptibility, or susceptibility, wherein the one or more marker locus is selected from the group consisting of: (a) 9.1, 9.2, 9.3; (b) a marker locus linked to a marker locus of (a); and (c) a marker locus localizing within a chromosome interval including a marker pair 9.1 and 9.3.
 6. The method claim 5, wherein the detecting comprises detecting at least one allelic form of a polymorphic simple sequence repeat (SSR) or a single nucleotide polymorphism (SNP).
 7. The method claim 5, wherein the detecting comprises amplifying the marker locus or a portion of the marker locus and detecting the resulting amplified marker amplicon.
 8. The method of claim 7, wherein the amplifying comprises employing a polymerase chain reaction (PCR) or ligase chain reaction (LCR) using a nucleic acid isolated from the first melon plant or germplasm as a template in the PCR or LCR.
 9. The method claim 5, wherein the at least one allele is a favorable allele that positively correlates with FOM race 1,2 resistance or improved FOM race 1,2 resistance. 